Michelle J. Wilkinson

Surgical treatment of gastrointestinal stromal tumour of the rectum in the era of imatinib

Gastrointestinal stromal tumours (GISTs) of the rectum often require radical surgery to achieve complete resection. This study investigated the management and outcome of surgery for rectal GISTs and the role of imatinib.

BRAF- and MEK-Targeted Small Molecule Inhibitors Exert Enhanced Antimelanoma Effects in Combination With Oncolytic Reovirus Through ER Stress

Reovirus type 3 (Dearing) (RT3D) infection is selective for cells harboring a mutated/activated RAS pathway. Therefore, in a panel of melanoma cell lines (including RAS mutant, BRAF mutant and RAS/BRAF wild-type), we assessed therapeutic combinations that enhance/suppress ERK1/2 signaling through use of BRAF/MEK inhibitors. In RAS mutant cells, the combination of RT3D with the BRAF inhibitor PLX4720 (paradoxically increasing ERK1/2 signaling in this context) did not enhance reoviral cytotoxicity. Instead, and somewhat surprisingly, RT3D and BRAF inhibition led to enhanced cell kill in BRAF mutated cell lines. Likewise, ERK1/2 inhibition, using the MEK inhibitor PD184352, in combination with RT3D resulted in enhanced cell kill in the entire panel. Interestingly, TCID50 assays showed that BRAF and MEK inhibitors did not affect viral replication. Instead, enhanced efficacy was mediated through ER stress-induced apoptosis, induced by the combination of ERK1/2 inhibition and reovirus infection. In vivo, combined treatments of RT3D and PLX4720 showed significantly increased activity in BRAF mutant tumors in both immune-deficient and immune-competent models. These data provide a strong rationale for clinical translation of strategies in which RT3D is combined with BRAF inhibitors (in BRAF mutant melanoma) and/or MEK inhibitors (in BRAF and RAS mutant melanoma).

Surgical resection for non-familial adenomatous polyposis-related intra-abdominal fibromatosis.

Intra‐abdominal fibromatosis (IAF) in the context of familial adenomatosis polyposis (FAP) is associated with significant morbidity and high recurrence rates after surgical resection. Non‐surgical treatments are therefore advocated. This study explored outcomes in patients with IAF not associated with FAP who underwent surgical resection.

Outcomes of routine ilioinguinal lymph node dissection for palpable inguinal melanoma nodal metastasis

Patients who present with palpable inguinal melanoma nodal metastasis have two surgical options: inguinal or ilioinguinal lymph node dissection. Indications for either operation remain controversial. This study examined survival and recurrence outcomes following ilioinguinal dissection for patients with palpable inguinal nodal metastasis, and assessed the incidence and preoperative predictors of pelvic nodal metastasis.

Oncolytic vaccinia virus combined with radiotherapy induces apoptotic cell death in sarcoma cells by down-regulating the inhibitors of apoptosis

Advanced extremity melanoma and sarcoma present a significant therapeutic challenge, requiring multimodality therapy to treat or even palliate disease. These aggressive tumours are relatively chemo-resistant, therefore new treatment approaches are urgently required. We have previously reported on the efficacy of oncolytic virotherapy (OV) delivered by isolated limb perfusion. In this report, we have improved therapeutic outcomes by combining OV with radiotherapy. In vitro, the combination of oncolytic vaccinia virus (GLV-1h68) and radiotherapy demonstrated synergistic cytotoxicity. This effect was not due to increased viral replication, but mediated through induction of intrinsic apoptosis. GLV-1h68 therapy downregulated the anti-apoptotic BCL-2 proteins (MCL-1 and BCL-XL) and the downstream inhibitors of apoptosis, resulting in cleavage of effector caspases 3 and 7. In an in vivo ILP model, the combination of OV and radiotherapy significantly delayed tumour growth and prolonged survival compared to single agent therapy. These data suggest that the virally-mediated down-regulation of anti-apoptotic proteins may increase the sensitivity of tumour cells to the cytotoxic effects of ionizing radiation. Oncolytic virotherapy represents an exciting candidate for clinical development when delivered by ILP. Its ability to overcome anti-apoptotic signals within tumour cells points the way to further development in combination with conventional anti-cancer therapies.

Isolated limb perfusion with melphalan, tumour necrosis factor-alpha and oncolytic vaccinia virus improves tumour targeting and prolongs survival in a rat model of advanced extremity sarcoma.

Isolated limb perfusion (ILP) is a treatment for advanced extremity sarcoma and in‐transit melanoma. Advancing this procedure by investigating the addition of novel agents, such as cancer‐selective oncolytic viruses, may improve both the therapeutic efficacy of ILP and the tumour‐targeted delivery of oncolytic virotherapy. Standard in vitro assays were used to characterise single agent and combinatorial activities of melphalan, tumour necrosis factor‐alpha (TNF‐α) and Lister strain vaccinia virus (GLV‐1h68) against BN175 rat sarcoma cells. An orthotopic model of advanced extremity sarcoma was used to evaluate survival of animals after ILP with combinations of TNF‐α, melphalan and GLV‐1h68. We investigated the efficiency of viral tumour delivery by ILP compared to intravenous therapy, the locoregional and systemic biodistribution of virus after ILP, and the effect of mode of administration on antibody response. The combination of melphalan and GLV‐1h68 was synergistic in vitro. The addition of virus to standard ILP regimens was well tolerated and demonstrated superior tumour targeting compared to intravenous administration. Triple therapy (melphalan/TNF‐α/GLV‐1h68) resulted in increased tumour growth delay and enhanced survival compared to other treatment regimens. Live virus was recovered in large amounts from perfused regions, but in smaller amounts from systemic organs. The addition of oncolytic vaccinia virus to existing TNF‐α/melphalan‐based ILP strategies results in survival advantage in an immunocompetent rat model of advanced extremity sarcoma. Virus administered by ILP has superior tumour targeting compared to intravenous delivery. Further evaluation and clinical translation of this approach is warranted.

Isolated limb perfusion with biochemotherapy and oncolytic virotherapy combines with radiotherapy and surgery to overcome treatment resistance in an animal model of extremity soft tissue sarcoma

The management of locally advanced or recurrent extremity sarcoma often necessitates multimodal therapy to preserve a limb, of which isolated limb perfusion (ILP) is a key component. However, with standard chemotherapeutic agents used in ILP, the duration of response is limited. Novel agents or treatment combinations are urgently needed to improve outcomes. Previous work in an animal model has demonstrated the efficacy of oncolytic virotherapy when delivered by ILP and, in this study, we report further improvements from combining ILP‐delivered oncolytic virotherapy with radiation and surgical resection. In vitro, the combination of radiation with an oncolytic vaccinia virus (GLV‐1h68) and melphalan demonstrated increased cytotoxicity in a panel of sarcoma cell lines. The effects were mediated through activation of the intrinsic apoptotic pathway. In vivo, combinations of radiation, oncolytic virotherapy and standard ILP resulted in delayed tumour growth and prolonged survival when compared with standard ILP alone. However, local disease control could only be secured when such treatment was combined with surgical resection, the timing of which was crucial in determining outcome. Combinations of oncolytic virotherapy with surgical resection and radiation have direct clinical relevance in extremity sarcoma and represent an exciting prospect for improving outcomes in this pathology.

Genetically modified lentiviruses that preserve microvascular function protect against late radiation damage in normal tissues

Vascular protection using gene therapy can reduce the late toxicities of radiotherapy. Skin protection from radiation goes viral With recent improvements in cancer therapy, an increasing number of people are living as cancer survivors, in many cases with long-term side effects caused by the cancer treatment. These effects include radiation-induced vascular dysfunction and fibrosis, which interfere with tissue reconstruction using skin flaps after mastectomy in breast cancer patients. Khan et al. developed a virus-based gene therapy approach to address this problem, up-regulating one gene to preserve skin flap volume and knocking down another to reduce radiation-induced skin contracture. The authors tested their approach in rat models of radiation therapy and skin flap reconstruction and also demonstrated that the gene therapy did not interfere with the anticancer effects of radiation. Improvements in cancer survival mean that long-term toxicities, which contribute to the morbidity of cancer survivorship, are being increasingly recognized. Late adverse effects (LAEs) in normal tissues after radiotherapy (RT) are characterized by vascular dysfunction and fibrosis causing volume loss and tissue contracture, for example, in the free flaps used for immediate breast reconstruction after mastectomy. We evaluated the efficacy of lentivirally delivered superoxide dismutase 2 (SOD2) overexpression and connective tissue growth factor (CTGF) knockdown by short hairpin RNA in reducing the severity of LAEs in an animal model of free flap LAEs. Vectors were delivered by intra-arterial injection, ex vivo, to target the vascular compartment. LVSOD2 and LVshCTGF monotherapy before irradiation resulted in preservation of flap volume or reduction in skin contracture, respectively. Flaps transduced with combination therapy experienced improvements in both volume loss and skin contracture. Both therapies reduced the fibrotic burden after irradiation. LAEs were associated with impaired vascular perfusion, loss of endothelial permeability, and stromal hypoxia, which were all reversed in the treatment model. Using a tumor recurrence model, we showed that SOD2 overexpression in normal tissues did not compromise the efficacy of RT against tumor cells but appeared to enhance it. LVSOD2 and LVshCTGF combination therapy by targeted, intravascular delivery reduced LAE severities in normal tissues without compromising the efficacy of RT and warrants translational evaluation as a free flap–targeted gene therapy.

Enhanced cytotoxicity of reovirus and radiotherapy in melanoma cells is mediated through increased viral replication and mitochondrial apoptotic signalling

Oncolytic viruses selectively target and replicate in cancer cells, providing us with a unique tool with which to target and kill tumour cells. These viruses come from a diverse range of viral families including reovirus type 3 Dearing (RT3D), a non-pathogenic human double-stranded RNA oncolytic virus, which has been shown to be an effective therapeutic agent, both as a mono-therapy and in combination with traditional chemotherapeutic drugs. This study investigated the interaction between RT3D and radiotherapy in melanoma cell lines with a BRAF mutant, Ras mutant or BRAF/Ras wild type genotype. The data indicates that RT3D combined with radiotherapy significantly increased cytotoxicity relative to either single agent, independent of genotype, both in vitro and in vivo. The mechanism of enhanced cytotoxicity was dependent on an increase in viral replication, mediated by CUG2 up-regulation and subsequent down-regulation of pPKR and p-eIF2α, leading to the activation of mitochondrial apoptotic signalling resulting in increased cell death.

Adenovirally delivered enzyme prodrug therapy with herpes simplex virus-thymidine kinase in composite tissue free flaps shows therapeutic efficacy in rat models of glioma.

Introduction: Free flap gene therapy exploits a novel therapeutic window when viral vectors can be delivered into a flap ex vivo. The authors investigated the therapeutic potential of an adenovirally-delivered thymidine kinase/ganciclovir prodrug system expressed following vector delivery into a free flap. Methods: The authors demonstrated direct in vitro cytotoxicity by treating a panel of malignant cell lines with the thymidine kinase/ganciclovir system and demonstrated significant cell kill proportional to the multiplicity of infection of adenoviral vector expressing thymidine kinase. Bystander cytotoxicity was demonstrated using conditioned media from producer cells (expressing adenovirally-delivered thymidine kinase and treated with ganciclovir) to demonstrate cytotoxicity in naive tumor cells. The authors investigated the effect of adenoviral vector expressing thymidine kinase/ganciclovir therapy in vivo, using models of microscopic and macroscopic residual disease in a rodent superficial inferior epigastric artery flap model. Results: The authors observed retardation of tumor volume growth in both microscopic (p = 0.0004) and macroscopic (p = 0.0005) residual disease models and prolongation of animal survival. Gene expression studies demonstrated that viral genomic material was found predominantly in flap tissues but declined over time. Conclusions: The authors describe the utility of virally delivered enzyme/prodrug therapy, using a free flap as a vehicle for delivery. They discuss the merits and limitations of this approach and the unique role of therapeutic free flaps among reconstructive techniques available to the plastic surgeon.