Kellie B. Haworth

Going back to class I: MHC and immunotherapies for childhood cancer

After decades of unfulfilled promise, immunotherapies for cancer have reached a tipping point, with several FDA approved products now on the market and many more showing promise in both adult and pediatric clinical trials. Tumor cell expression of MHC class I has emerged as a potential determinant of the therapeutic success of many immunotherapy approaches. Here we review current knowledge regarding MHC class I expression in pediatric cancers including a discussion of prognostic significance, the opposing influence of MHC on T‐cell versus NK‐mediated therapies, and strategies to reverse or circumvent MHC down‐regulation.Pediatr Blood Cancer 2015;62:571–576.

To Infection and Beyond: The Multi-Pronged Anti-Cancer Mechanisms of Oncolytic Viruses

Over the past 1–2 decades we have witnessed a resurgence of efforts to therapeutically exploit the attributes of lytic viruses to infect and kill tumor cells while sparing normal cells. We now appreciate that the utility of viruses for treating cancer extends far beyond lytic cell death. Viruses are also capable of eliciting humoral and cellular innate and adaptive immune responses that may be directed not only at virus-infected cells but also at uninfected cancer cells. Here we review our current understanding of this bystander effect, and divide the mechanisms into lytic, cytokine, innate cellular, and adaptive phases. Knowing the key pathways and molecular players during virus infection in the context of the cancer microenvironment will be critical to devise strategies to maximize the therapeutic effects of oncolytic viroimmunotherapy.

Oncolytic HSV virotherapy in murine sarcomas differentially triggers an antitumor T-cell response in the absence of virus permissivity

Multiple studies have indicated that in addition to direct oncolysis, virotherapy promotes an antitumor cytotoxic T cell response important for efficacy. To study this phenomenon further, we tested three syngeneic murine sarcoma models that displayed varied degrees of permissiveness to oncolytic herpes simplex virus replication and cytotoxicity in vitro, with the most permissive being comparable to some human sarcoma tumor lines. The in vivo antitumor effect ranged from no or modest response to complete tumor regression and protection from tumor rechallenge. The in vitro permissiveness to viral oncolysis was not predictive of the in vivo antitumor effect, as all three tumors showed intact interferon signaling and minimal permissiveness to virus in vivo. Tumor shrinkage was T-cell mediated with a tumor-specific antigen response required for maximal antitumor activity. Further analysis of the innate and adaptive immune microenvironment revealed potential correlates of susceptibility and resistance, including favorable and unfavorable cytokine profiles, differential composition of intratumoral myeloid cells, and baseline differences in tumor cell immunogenicity and tumor-infiltrating T-cell subsets. It is likely that a more complete understanding of the interplay between the immunologic immune microenvironment and virus infection will be necessary to fully leverage the antitumor effects of this therapeutic platform.

Pediatric cancer gone viral. Part I: strategies for utilizing oncolytic herpes simplex virus-1 in children

Progress for improving outcomes in pediatric patients with solid tumors remains slow. In addition, currently available therapies are fraught with numerous side effects, often causing significant life-long morbidity for long-term survivors. The use of viruses to kill tumor cells based on their increased vulnerability to infection is gaining traction, with several viruses moving through early and advanced phase clinical testing. The prospect of increased efficacy and decreased toxicity with these agents is thus attractive for pediatric cancer. In part I of this two-part review, we focus on strategies for utilizing oncolytic engineered herpes simplex virus (HSV) to target pediatric malignancies. We discuss mechanisms of action, routes of delivery, and the role of preexisting immunity on antitumor efficacy. Challenges to maximizing oncolytic HSV in children are examined, and we highlight how these may be overcome through various arming strategies. We review the preclinical and clinical evidence demonstrating safety of a variety of oncolytic HSVs. In Part II, we focus on the antitumor efficacy of oncolytic HSV in pediatric tumor types, pediatric clinical advances made to date, and future prospects for utilizing HSV in pediatric patients with solid tumors.

Characterization of MHC Class I and β-2-Microglobulin Expression in Pediatric Solid Malignancies to Guide Selection of Immune-Based Therapeutic Trials.

Over 10,000 US children are diagnosed with cancer yearly. Though outcomes have improved by optimizing conventional therapies, recent immunotherapeutic successes in adult cancers are emerging. Cytotoxic T lymphocytes (CTLs) are the primary executioners of adaptive antitumor immunity and require antigenic presentation in the context of major histocompatibility complex (MHC) class I and the associated β‐2‐microglobulin (B2M). Loss of MHC I expression is a common immune escape mechanism in adult malignancies, but pediatric cancers have not been thoroughly characterized. The essential nature of MHC I expression in CTL‐mediated cell death may dictate the success of immunotherapies, which rely on eliciting an adaptive response.

Pediatric cancer gone viral. Part II: potential clinical application of oncolytic herpes simplex virus-1 in children

Oncolytic engineered herpes simplex viruses (HSVs) possess many biologic and functional attributes that support their use in clinical trials in children with solid tumors. Tumor cells, in an effort to escape regulatory mechanisms that would impair their growth and progression, have removed many mechanisms that would have protected them from virus infection and eventual virus-mediated destruction. Viruses engineered to exploit this weakness, like mutant HSV, can be safely employed as tumor cell killers, since normal cells retain these antiviral strategies. Many preclinical studies and early phase trials in adults demonstrated that oncolytic HSV can be safely used and are highly effective in killing tumor cells that comprise pediatric malignancies, without generating the toxic side effects of nondiscriminatory chemotherapy or radiation therapy. A variety of engineered viruses have been developed and tested in numerous preclinical models of pediatric cancers and initial trials in patients are underway. In Part II of this review series, we examine the preclinical evidence to support the further advancement of oncolytic HSV in the pediatric population. We discuss clinical advances made to date in this emerging era of oncolytic virotherapy.

Immune profiling of NF1-associated tumors reveals histologic subtype distinctions and heterogeneity: Implications for immunotherapy

Successful treatment of neurofibromatosis type 1 (NF1)-associated tumors poses a significant clinical challenge. While the primary underlying genetic defect driving RAS signaling is well described, recent evidence suggests immune dysfunction contributes to tumor pathogenesis and malignant transformation. As immunologic characterizations, prognostic and predictive of immunotherapeutic clinical response in other cancers, are not fully described for benign and malignant NF1-related tumors, we sought to define their immunologic profiles. We determined the expression of human leukocyte antigen (HLA)-A/-B/-C, β-2-microglobulin (B2M), and T cell inhibitory ligands PD-L1 and CTLA-4 by microarray gene analysis and flow cytometry. We examined HLA-A/-B/-C, B2M, and PD-L1 expression on thirty-six NF1-associated tumor samples by immunohistochemistry, and correlated these with tumoral CD4+, CD8+, FOXP3+, CD56+, and CD45RO+ lymphocytic infiltrates. We evaluated several tumors from a single patient, observing trends of increasing immunogenicity over time, even with disease progression. We observed similarly immunogenic profiles for malignant peripheral nerve sheath tumors (MPNST) and nodular and plexiform neurofibromas, contrasting with diffuse neurofibromas. These studies suggest that while immunotherapies may offer some benefit for MPNST and nodular and plexiform neurofibromas, tumor heterogeneity might pose a significant clinical challenge to this novel therapeutic approach.Successful treatment of neurofibromatosis type 1 (NF1)-associated tumors poses a significant clinical challenge. While the primary underlying genetic defect driving RAS signaling is well described, recent evidence suggests immune dysfunction contributes to tumor pathogenesis and malignant transformation. As immunologic characterizations, prognostic and predictive of immunotherapeutic clinical response in other cancers, are not fully described for benign and malignant NF1-related tumors, we sought to define their immunologic profiles. We determined the expression of human leukocyte antigen (HLA)-A/-B/-C, β-2-microglobulin (B2M), and T cell inhibitory ligands PD-L1 and CTLA-4 by microarray gene analysis and flow cytometry. We examined HLA-A/-B/-C, B2M, and PD-L1 expression on thirty-six NF1-associated tumor samples by immunohistochemistry, and correlated these with tumoral CD4+, CD8+, FOXP3+, CD56+, and CD45RO+ lymphocytic infiltrates. We evaluated several tumors from a single patient, observing trends of increasing immunogenicity over time, even with disease progression. We observed similarly immunogenic profiles for malignant peripheral nerve sheath tumors (MPNST) and nodular and plexiform neurofibromas, contrasting with diffuse neurofibromas. These studies suggest that while immunotherapies may offer some benefit for MPNST and nodular and plexiform neurofibromas, tumor heterogeneity might pose a significant clinical challenge to this novel therapeutic approach.

Oncolytic Herpes Virus rRp450 Shows Efficacy in Orthotopic Xenograft Group 3/4 Medulloblastomas and Atypical Teratoid/Rhabdoid Tumors

Pediatric brain tumors including medulloblastoma and atypical teratoid/rhabdoid tumor are associated with significant mortality and treatment-associated morbidity. While medulloblastoma tumors within molecular subgroups 3 and 4 have a propensity to metastasize, atypical teratoid/rhabdoid tumors frequently afflict a very young patient population. Adjuvant treatment options for children suffering with these tumors are not only sub-optimal but also associated with many neurocognitive obstacles. A potentially novel treatment approach is oncolytic virotherapy, a developing therapeutic platform currently in early-phase clinical trials for pediatric brain tumors and recently US Food and Drug Administration (FDA)-approved to treat melanoma in adults. We evaluated the therapeutic potential of the clinically available oncolytic herpes simplex vector rRp450 in cell lines derived from medulloblastoma and atypical teratoid/rhabdoid tumor. Cells of both tumor types were supportive of virus replication and virus-mediated cytotoxicity. Orthotopic xenograft models of medulloblastoma and atypical teratoid/rhabdoid tumors displayed significantly prolonged survival following a single, stereotactic intratumoral injection of rRp450. Furthermore, addition of the chemotherapeutic prodrug cyclophosphamide (CPA) enhanced rRp450’s in vivo efficacy. In conclusion, oncolytic herpes viruses with the ability to bioactivate the prodrug CPA within the tumor microenvironment warrant further investigation as a potential therapy for pediatric brain tumors.

Immune profiles of desmoplastic small round cell tumor and synovial sarcoma suggest different immunotherapeutic susceptibility upfront compared to relapse specimens

Desmoplastic small round cell tumor (DSRCT) and synovial sarcoma are rare tumors with dismal outcomes requiring new therapeutic strategies. Immunotherapies have shown promise in several cancer types, but have not been evaluated in DSRCT and synovial sarcoma. Because the immune microenvironment can provide indications of the inflammatory nature of tumors, immunohistochemical staining is able to assess the tumor immune infiltrates in both tumor types.

Abstract A14: Checkpoinblockade enhances oncolytic herpes virotherapy in immunosuppressive sarcoma models

Most solid tumors are characterized by an immunosuppressive microenvironment, limiting the effectiveness of antitumor immunotherapeutics. Programmed cell death protein (PD)-1-mediated T cell suppression via engagement of its ligand, PD-L1, is of particular interest due to recent successes in selected adult cancers. The utility of PD1-directed therapy for pediatric cancers is unknown, especially given the paucity of mutations and thus infrequent neoantigens in many types of childhood tumors. Oncolytic virotherapy induces tumor shrinkage via a multistep process including direct tumor cell lysis, induction of cytotoxic or apoptosis-sensitizing cytokines, and induction of anti-tumor T cell responses. We recently demonstrated that intratumoral injection of an oncolytic herpes virus induced growth delays and in some cases durable remissions in several mouse models of rhabdomyosarcoma. The effects were T cell mediated, rendering surviving mice resistant to tumor rechallenge and being absent in athymic nude hosts (Leddon et al., Mol Ther-Oncolytics 1, Article number: 14010, 2015). We found these tumor models express PD-L1, suggesting that T cell checkpoints may in part limit virus-induced antitumor immunity. Here we show the implantable C57Bl/6 rhabdomyosarcoma model, M3-9-M, showed a moderate response to single-agent Seprehvir (HSV1716), a virus currently in pediatric clinical trials (NCT00931931, NCT02031965). Single-agent PD-1 blockade also showed moderate but significant tumor growth delay with no complete responses. Strikingly, combining these two therapies together substantially prolonged overall survival with several complete responses post 40 days treatment. We hypothesize that virotherapy-induced chemokine secretion increases the recruitment of antitumor T cells, while PD-1 blockade increases their effectiveness. Overall, our data suggest the combination of PD-1 and oncolytic herpes virotherapy may be an effective treatment strategy for some cancers. Citation Format: Chun-yu Chun, Pin-Yi Wang, Brian Hutzen, Kellie Haworth, Joe Conner, Timothy Cripe. Checkpoinblockade enhances oncolytic herpes virotherapy in immunosuppressive sarcoma models. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A14.