PD-1 Blockade Following Isolated Limb Perfusion with Vaccinia Virus Prevents Local and Distant Relapse of Soft-tissue Sarcoma.

PURPOSE: The prevention and treatment of metastatic sarcoma are areas of significant unmet need. Immune checkpoint inhibitor monotherapy has shown little activity in sarcoma and there is great interest in identifying novel treatment combinations that may augment responses. In vitro and in vivo, we investigated the potential for an oncolytic vaccinia virus (GLV-1h68) delivered using isolated limb perfusion (ILP) to promote antitumor immune responses and augment response to PD-1 blockade in sarcoma.Experimental Design: In an established animal model of extremity sarcoma, we evaluated the potential of locoregional delivery of a vaccinia virus (GLV-1h68) alongside biochemotherapy (melphalan/TNFα) in ILP. Complementary in vitro assays for markers of immunogenic cell death were performed in sarcoma cell lines. RESULTS: PD-1 monotherapy had minimal efficacy in vivo, mimicking the clinical scenario. Pretreatment with GLV-1h68 delivered by ILP (viral ILP) significantly improved responses. Furthermore, when performed prior to surgery and radiotherapy, viral ILP and PD-1 blockade prevented both local and distant relapse, curing a previously treatment-refractory model. Enhanced therapy was associated with marked modulation of the tumor microenvironment, with an increase in the number and penetrance of intratumoral CD8+ T cells and expansion and activation of dendritic cells. GLV-1h68 was capable of inducing markers of immunogenic cell death in human sarcoma cell lines. CONCLUSIONS: Viral ILP augments the response to PD-1 blockade, transforming this locoregional therapy into a potentially effective systemic treatment for sarcoma and warrants translational evaluation.

ATR Inhibition Potentiates the Radiation-induced Inflammatory Tumor Microenvironment.

PURPOSE: ATR inhibitors (ATRi) are in early phase clinical trials and have been shown to sensitize to chemotherapy and radiotherapy preclinically. Limited data have been published about the effect of these drugs on the tumor microenvironment.Experimental Design: We used an immunocompetent mouse model of HPV-driven malignancies to investigate the ATR inhibitor AZD6738 in combination with fractionated radiation (RT). Gene expression analysis and flow cytometry were performed posttherapy. RESULTS: Significant radiosensitization to RT by ATRi was observed alongside a marked increase in immune cell infiltration. We identified increased numbers of CD3+ and NK cells, but most of this infiltrate was composed of myeloid cells. ATRi plus radiation produced a gene expression signature matching a type I/II IFN response, with upregulation of genes playing a role in nucleic acid sensing. Increased MHC I levels were observed on tumor cells, with transcript-level data indicating increased antigen processing and presentation within the tumor. Significant modulation of cytokine gene expression (particularly CCL2, CCL5, and CXCL10) was found in vivo, with in vitro data indicating CCL3, CCL5, and CXCL10 are produced from tumor cells after ATRi + RT. CONCLUSIONS: We show that DNA damage by ATRi and RT leads to an IFN response through activation of nucleic acid-sensing pathways. This triggers increased antigen presentation and innate immune cell infiltration. Further understanding of the effect of this combination on the immune response may allow modulation of these effects to maximize tumor control through antitumor immunity.

Combined ATR and DNA-PK Inhibition Radiosensitizes Tumor Cells Independently of Their p53 Status.

Head and neck squamous cell carcinoma (HNSCC) is a significant cause of cancer deaths. Cisplatin-based chemoradiotherapy is a standard of care for locally advanced disease. ATR and DNA-PK inhibition (DNA-PKi) are actively being investigated in clinical trials with preclinical data supporting clinical translation as radiosensitizers. Here, we hypothesized that targeting both ATR and DNA-PK with small molecule inhibitors would increase radiosensitization of HNSCC cell lines. Radiosensitization was assessed by Bliss independence analysis of colony survival data. Strong cell cycle perturbing effects were observed with ATR inhibition reversing the G2/M arrest observed for radiation-DNA-PKi. Increased apoptosis in combination groups was measured by Sub-G1 DNA populations. DNA-PKi increased radiation-induced RAD51 and gamma-H2Ax foci, with the addition of ATR inhibition reducing levels of both. A sharp increase in nuclear fragmentation after aberrant mitotic transit appears to be the main driver of decreased survival due to irradiation and dual ATR/DNA-PKi. Dual inhibition of DNA-PK and ATR represents a novel approach in combination with radiation, with efficacy appearing to be independent of p53 status. Due to toxicity concerns, careful assessment is necessary in any future translation of single or dual radiosensitization approaches. Ongoing clinical trials into the ATR inhibitor AZD6738 plus radiation, and the phenotypically similar combination of AZD6738 and the PARP inhibitor olaparib, are likely to be key in ascertaining the toxicity profile of such combinations.

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

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.

The tumour microenvironment after radiotherapy: mechanisms of resistance and recurrence.

Radiotherapy plays a central part in curing cancer. For decades, most research on improving treatment outcomes has focused on modulating radiation-induced biological effects on cancer cells. Recently, we have better understood that components within the tumour microenvironment have pivotal roles in determining treatment outcomes. In this Review, we describe vascular, stromal and immunological changes that are induced in the tumour microenvironment by irradiation and discuss how these changes may promote radioresistance and tumour recurrence. We also highlight how this knowledge is guiding the development of new treatment paradigms in which biologically targeted agents will be combined with radiotherapy.

Donor site morbidity of the medial plantar artery flap studied with gait and pressure analysis.

BACKGROUND: The medial plantar artery flap (MPA) allows transfer of both glabrous (smooth and free from hair) and sensate tissue. It has been suggested that the non-weight bearing instep area of the foot provides tissue for transfer with minimal donor morbidity. However the abductor hallucis muscle and plantar fascia are dissected during flap harvest which may affect foot mechanics. METHODS: Patients were included who had undergone MPA flap harvest and were walking unaided. The majority of the patients studied had problems with soft tissues of their heels rather than trauma as a starting point. Laboratory normals and the patient's contralateral limb were used as controls. Gait and pressure analysis were performed using 3D gait analysis and high resolution pressure analysis. RESULTS: This study included 6 patients, with 5 chronic wounds (4 ipsilateral, 1 contralateral) and 1 traumatic ankle defect. QUESTIONNAIRE RESULTS: Enneking scores: 67.9% return to function; Foot Function Index scores: 39.1% loss of function. GAIT ANALYSIS: Significant differences were seen in kinetic and kinematic data. PRESSURE ANALYSIS: The donor site group had significantly less pressure in the great toe (38.1kPa vs. 78.1kPa, p=0.013), significantly slower transition through the midfoot (445.2ms vs. 352.07ms, p=0.016) and increased impulse in the heel (3.1kPa/s vs. 11.7kPa/s, p=0.038). CONCLUSIONS: This study demonstrates subjective and objective evidence of MPA donor site morbidity. Comparison to other studies looking at gait and pressure changes seen after flap reconstruction of the plantar region suggest that much of this difference may be attributable to ipsilateral reconstruction. As the majority had chronic problems with the soft tissues over the heel some of these biomechanical responses could be related to learned behaviour preoperatively or continued discomfort in the heel pad. Nonetheless it demonstrates accurately the effect of the technique overall on the function of the foot. The changes in the region of the great toe may be solely attributable to MPA harvest. These results suggest that MPA harvest is not free of donor morbidity.

Surgery for a tree surgeon? Acute presentation of contact dermatitis due to Ailanthus altissima.

A tree surgeon presented to hospital with multiple blackening, non-blanching regions of skin on both forearms, following exposure to sap from the 'tree of heaven' (Ailanthus altissima). A referral to plastic surgery was made to consider debridement. Following input from the national poisons centre and dermatology, conservative management with emollient was undertaken. The lesions blistered and exfoliated and were treated with topical steroid and oral antihistamines. Resolving erythema was the only symptom at three weeks. A. altissima, also known as the 'tree of heaven' has known toxins in its bark, leaves and flowers but is also commonly used in folk medicine. Two previous cases of contact dermatitis are reported in the literature but not with acute photo documentation of the lesions or with surgical referral. This demonstrates an important lesson that debridement would not be the appropriate management despite the initial presentation.

Fasciocutaneous flaps of the subscapular artery axis to reconstruct large extremity defects.

INTRODUCTION: The scapular, parascapular and thoracodorsal artery perforator (TDAP) flaps represent fasciocutaneous flaps derived from the subscapular artery axis. These flaps can be harvested individually or combined as conjoint flaps, tailored to reconstruct a wide variety of defects in the extremities. ANALYSIS AND METHODS: All patients undergoing free-flap reconstruction at North Bristol trust with a fasciocutaneous flap of the subscapular axis from April 2006 until April 2010 were included. This cohort of 45 patients was retrospectively analysed. The Enneking score for return of limb function was used as an outcome measure after reconstruction. Donor-site morbidity analysis was carried out prospectively using Oxford Medical Research Council (MRC) score, Vancouver Scar Scale and disability of arm, shoulder and hand questionnaire (DASH) scores. RESULTS: A total of 45 patients had extremity reconstruction using flaps of the subscapular artery axis following severe limb trauma, often comprising open tibial fractures. A total of 42 patients had lower limb injuries and three had upper limb injuries. All flaps survived. The mean Injury Severity Score (ISS) was 9.3, the mean Enneking score was 27 at 12 months mean follow-up. In the nine conjoint flaps, the mean area of tissue resurfaced was 257 cm2. CONCLUSIONS: In this case series of fasciocutaneous flaps of the subscapular artery axis, we establish that these flaps are robust and versatile. They replace 'like-with-like' and have good patient satisfaction. The donor site can be closed primarily, is discrete and has minimal donor morbidity. The conjoint flaps can be used for reconstruction of very large defects without the need to sacrifice functionally important muscle.