Joseph L. Benci

Radiation and dual checkpoint blockade activate non-redundant immune mechanisms in cancer

Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.

Mitotic progression following DNA damage enables pattern recognition within micronuclei

Inflammatory gene expression following genotoxic cancer therapy is well documented, yet the events underlying its induction remain poorly understood. Inflammatory cytokines modify the tumour microenvironment by recruiting immune cells and are critical for both local and systemic (abscopal) tumour responses to radiotherapy. A poorly understood feature of these responses is the delayed onset (days), in contrast to the acute DNA-damage responses that occur in minutes to hours. Such dichotomous kinetics implicate additional rate-limiting steps that are essential for DNA-damage-induced inflammation. Here we show that cell cycle progression through mitosis following double-stranded DNA breaks leads to the formation of micronuclei, which precede activation of inflammatory signalling and are a repository for the pattern-recognition receptor cyclic GMP–AMP synthase (cGAS). Inhibiting progression through mitosis or loss of pattern recognition by stimulator of interferon genes (STING)–cGAS impaired interferon signalling. Moreover, STING loss prevented the regression of abscopal tumours in the context of ionizing radiation and immune checkpoint blockade in vivo. These findings implicate temporal modulation of the cell cycle as an important consideration in the context of therapeutic strategies that combine genotoxic agents with immune checkpoint blockade.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

18F-Fluorothymidine-Pet Imaging of Glioblastoma Multiforme: Effects of Radiation Therapy on Radiotracer Uptake and Molecular Biomarker Patterns

Introduction. PET imaging is a useful clinical tool for studying tumor progression and treatment effects. Conventional 18F-FDG-PET imaging is of limited usefulness for imaging Glioblastoma Multiforme (GBM) due to high levels of glucose uptake by normal brain and the resultant signal-to-noise intensity. 18F-Fluorothymidine (FLT) in contrast has shown promise for imaging GBM, as thymidine is taken up preferentially by proliferating cells. These studies were undertaken to investigate the effectiveness of 18F-FLT-PET in a GBM mouse model, especially after radiation therapy (RT), and its correlation with useful biomarkers, including proliferation and DNA damage. Methods. Nude/athymic mice with human GBM orthografts were assessed by microPET imaging with 18F-FDG and 18F-FLT. Patterns of tumor PET imaging were then compared to immunohistochemistry and immunofluorescence for markers of proliferation (Ki-67), DNA damage and repair (γH2AX), hypoxia (HIF-1α), and angiogenesis (VEGF). Results. We confirmed that 18F-FLT-PET uptake is limited in healthy mice but enhanced in the intracranial tumors. Our data further demonstrate that 18F-FLT-PET imaging usefully reflects the inhibition of tumor by RT and correlates with changes in biomarker expression. Conclusions. 18F-FLT-PET imaging is a promising tumor imaging modality for GBM, including assessing RT effects and biologically relevant biomarkers.

PIM kinases as therapeutic targets against advanced melanoma

Therapeutic strategies for the treatment of metastatic melanoma show encouraging results in the clinic; however, not all patients respond equally and tumor resistance still poses a challenge. To identify novel therapeutic targets for melanoma, we screened a panel of structurally diverse organometallic inhibitors against human-derived normal and melanoma cells. We observed that a compound that targets PIM kinases (a family of Ser/Thr kinases) preferentially inhibited melanoma cell proliferation, invasion, and viability in adherent and three-dimensional (3D) melanoma models. Assessment of tumor tissue from melanoma patients showed that PIM kinases are expressed in pre- and post-treatment tumors, suggesting PIM kinases as promising targets in the clinic. Using knockdown studies, we showed that PIM1 contributes to melanoma cell proliferation and tumor growth in vivo; however, the presence of PIM2 and PIM3 could also influence the outcome. The inhibition of all PIM isoforms using SGI-1776 (a clinically-available PIM inhibitor) reduced melanoma proliferation and survival in preclinical models of melanoma. This was potentiated in the presence of the BRAF inhibitor PLX4720 and in the presence of PI3K inhibitors. Our findings suggest that PIM inhibitors provide promising additions to the targeted therapies available to melanoma patients.

Abstract A056: Mechanisms of tumor response and resistance to radiation and dual checkpoint blockade in mice and patients

Immune checkpoint inhibitors result in impressive clinical responses but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here, we report major tumor regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation (RT) on a phase one clinical trial and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumors, resistance was common. Computational analysis of genome-wide and immune profiles of mice revealed resistance was due to T cell exhaustion driven by intrinsic and adaptive resistance through STAT1-mediated upregulation of PD-L1 on melanoma cells and tumor macrophages. Accordingly, optimal response in melanoma and other cancer types requires RT, anti-CTLA4, and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T regulatory cells (Tregs) to increase the CD8 T cell to Treg (CD8/Treg) ratio. RT promotes the infiltration of intratumoral antigen-specific CD8 T cells and enhances the diversity of their T cell receptor (TCR) repertoire. Together, anti-CTLA4 promotes expansion of T cells, while RT shapes the TCR repertoire of the expanded peripheral clones in a manner consistent with antigen-driven selection. Addition of PD-L1 blockade reverses T cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligo-clonal T cell expansion. Similar to results from mice, patients on our clinical trial with tumors showing high PD-L1 did not respond to RT + anti-CTLA4, demonstrated persistent T cell exhaustion, and rapidly progressed. In contrast, patients with low PD-L1 on melanoma cells or macrophages had markedly improved survival, with the best survival observed among those patients with low PD-L1 on both cell types. Thus, our results suggest that 1) RT can enhance response to anti-CTLA4 when the TCR and/or antigen repertoire are sub-optimal, 2) upregulation of PD-L1 through intrinsic and STAT1-mediated adaptive resistance mechanisms inhibits response to anti-CTLA4-based therapy unless PD-L1/PD-1 is blocked, and 3) the combination of RT, anti-CTLA4, and anti-PD-L1 promotes response and immunity through distinct mechanisms. Finally, although PD-L1 was a dominant resistance mechanism in our models, PD-L1-independent resistance mechanisms were also present and targetable. The next generation of clinical trials based on these findings are underway. Citation Format: Christina Twyman-Saint Victor, Andrew Rech, Amit Maity, Ramesh Rengan, Kristen Pauken, Erietta Stelekati, Joseph Benci, Bihui Xu, Hannah Dada, Pamela Odorizzi, Ramin Herati, Kathleen Mansfield, Dana Patsch, Ravi Amaravadi, Lynn Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, Xiaowei Xu, Michael Feldman, Tara Gangadhar, Stephen Hahn, E. John Wherry, Robert Vonderheide, Andy Minn. Mechanisms of tumor response and resistance to radiation and dual checkpoint blockade in mice and patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A056.

Identifying barriers to cancer survivors sharing their survivorship care plans with their healthcare provider

PurposeTo understand what factors influence whether a cancer survivor will share their survivorship care plan (SCP) with their healthcare provider (HCP).MethodsWe used data from 3231 cancer survivors who utilized the OncoLink SCP resource between 2009 and 2016. Random forest and stepwise regression were used to identify predictors of SCP satisfaction and barriers to survivors sharing their care plans with their HCPs.ResultsEighty-seven percent of users rated their satisfaction with their SCP as good or better; however, only 70% of survivors planned to share their SCP with their HCP. The most commonly reported reason for not sharing was a feeling that their HCP would not care. Self-reported satisfaction with their SCP was strongest predictor of whether a survivor would share their SCP. Gender, cancer status, number of chemotherapies received, and who was managing their healthcare were all associated with self-reported survivor satisfaction with their SCP.ConclusionsSurvivor satisfaction with SCPs was high, but there was a disconnect in the number of satisfied survivors and the number of survivors planning to share their SCP with their HCP. To bridge this gap, additional prompts that HCPs are expecting this information should be added to the care plans.Implications for Cancer SurvivorsOne of the primary functions of survivorship care plans is to improve communication between survivor and healthcare provider. While survivors are overwhelmingly satisfied with their SCP, additional steps are necessary to get survivors to share their SCP with their HCP.

Survivorship care planning in skin cancer: An unbiased statistical approach to identifying patterns of care-plan use

Nearly 1 in 5 Americans will develop skin cancer, and as a result, survivors of skin cancer compose one of the largest groups of cancer survivors. Survivorship care plans (SCPs) are an important tool for improving patient outcomes and provide critical information to both survivors and health care professionals. Recent efforts have been made to expand SCP utilization; however, which patients currently receive SCPs is poorly understood.

Abstract PR05: Mechanisms of tumor response and resistance to radiation and dual checkpoint blockade in mice and patients

Immune checkpoint inhibitors result in impressive clinical responses but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here, we report major tumor regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation (RT) on a phase one clinical trial and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumors, resistance was common. Computational analysis of genome-wide and immune profiles of mice revealed resistance was due to T cell exhaustion driven by adaptive resistance and prolonged interferon-gamma exposure, resulting in STAT1-mediated upregulation of PD-L1 on melanoma cells and tumor macrophages. Accordingly, optimal response in melanoma and other cancer types requires RT, anti-CTLA4, and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T regulatory cells, and macrophage depletion and/or PD-L1 blockade reverses T cell exhaustion. RT promotes the infiltration of intratumoral antigen-specific CD8 T cells and enhances the diversity of the T cell receptor (TCR) repertoire. RT with dual checkpoint blockade shapes the TCR repertoire of the expanded peripheral clones in a manner consistent with antigen-driven selection. Similar to results from mice, patients on our clinical trial with tumors showing high PD-L1 did not respond to RT + anti-CTLA4, demonstrated persistent T cell exhaustion, and rapidly progressed. In contrast, patients with low PD-L1 on melanoma cells or macrophages had markedly improved survival, with the best survival observed among those patients with low PD-L1 on both cell types. Thus, our results suggest that 1) RT can enhance response to anti-CTLA4 when the TCR and/or antigen repertoire are sub-optimal, 2) upregulation of PD-L1 through STAT1-mediated adaptive resistance mechanisms inhibits response to anti-CTLA4-based therapy unless PD-L1/PD-1 is blocked, and 3) the combination of RT, anti-CTLA4, and anti-PD-L1 promotes response and immunity through distinct mechanisms. Finally, although PD-L1 was a dominant resistance mechanism in our models, PD-L1-independent resistance mechanisms were also evident. The next generation of clinical trials based on these findings are underway. Citation Format: Christina Twyman-Saint Victor, Andrew Rech, Joseph Benci, Amit Maity, Ramesh Rengan, Kristen Pauken, Erietta Stelekati, Bihui Xu, Hannah Dada, Pamela Odorizzi, Ramin Herati, Ravi Amaravadi, Lynn Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, Xiaowei Xu, Michael Feldman, Tara Gangadhar, Steve Hahn, John Wherry, Robert Vonderheide, Andy Minn. Mechanisms of tumor response and resistance to radiation and dual checkpoint blockade in mice and patients. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR05.

Abstract 2858: Radiation and dual immune checkpoint blockade overcome tumor resistance and distinctly improve immunity

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Optimal results with immune checkpoint inhibitors such as CTLA4 and PD-1 will likely require combination therapy. This raises important questions about tumor resistance and non-redundant mechanisms of action. Pre-clinical and clinical data indicate that radiation (RT) may augment responses to immune checkpoint inhibition. We therefore evaluated this combination for metastatic melanoma using parallel studies in mice and humans. In a phase I clinical trial with 19 patients with multiple melanoma metastases, a single index lesion was irradiated with hypofractionated RT, delivered over two or three fractions, followed by four cycles of the anti-CTLA4 antibody ipilimumab. We reproduced this therapy in mice using the melanoma cell line B16-F10. For this, each flank of C57BL/6 mice was implanted with tumors to model multiple metastases. Mice received anti-CTLA4 (on days 5, 8, and 11), irradiation of one tumor using an image-guided micro-irradiator (20 Gy x 1 on day 8), or both treatments. Mechanistic studies were performed on material obtained from patients and mice at baseline and thereafter. Overall, treatment in the phase I study was well tolerated and toxicity was similar to that reported for anti-CTLA4. Major tumor regressions were observed in a subset of patients with metastatic melanoma treated with anti-CTLA4 + RT. In mice, although combined treatment enhanced the CD8 T cell to Treg ratios and improved responses in irradiated and unirradiated tumors, resistance was common. Genome-wide and unbiased analyses revealed that resistant tumors have increased PD-L1, interferon-stimulated genes, and exhausted T cells that depress the CD8/Treg ratio. Patients and mice with high PD-L1 tumors that were treated with RT + anti-CTLA4 poorly reinvigorated exhausted T cells, did not respond, and rapidly progressed. In mice, adding anti-PD-L1/PD-1 to RT + anti-CTLA4 reinvigorated exhausted T cells, leading to complete responses and immunity across multiple cancer types. The extent of T cell exhaustion/reinvigoration predicts response and can be assessed through peripheral blood. Resistance to RT + anti-CTLA4 results from depression in the CD8/Treg ratio due to elevated tumor PD-L1 and persistent T cell exhaustion. Both clinical and pre-clinical data suggest that the combination of RT with CTLA4 and PD-1 checkpoint blockade is a rational, non-redundant approach to overcoming tumor resistance and improving immunity in multiple cancer types. Citation Format: Andrew J. Rech, Christina Twyman-Saint Victor, Amit Maity, Ramesh Rengan, Kristen E. Pauken, Erietta Stelekati, Joseph Benci, Bihui Xu, Hannah Dada, Pamela M. Odorizzi, Ramin S. Herati, Kathleen D. Mansfield, Dana Patsch, Ravi K. Amaravadi, Lynn M. Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, Xiaowei Xu, Michael D. Feldman, Tara C. Gangadhar, Stephen M. Hahn, E. John Wherry, Andy J. Minn, Robert H. Vonderheide. Radiation and dual immune checkpoint blockade overcome tumor resistance and distinctly improve immunity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2858. doi:10.1158/1538-7445.AM2015-2858