Safety culture and incident learning systems in radiation oncology: Staff perceptions across Australia and New Zealand.

INTRODUCTION: Radiation therapy has a highly complex pathway and uses detailed quality assurance protocols and incident learning systems (ILSs) to mitigate risk; however, errors can still occur. The safety culture (SC) in a department influences its commitment and effectiveness in maintaining patient safety. METHODS: Perceptions of SC and knowledge and understanding of ILSs and their use were evaluated for radiation oncology staff across Australia and New Zealand (ANZ). A validated healthcare survey tool (the Hospital Survey on Patient Safety Culture) was used, with additional specialty-focussed supporting questions. A total of 220 radiation oncologists, radiation therapists and radiation oncology medical physicists participated. RESULTS: An overall positive SC was indicated, with strength in teamwork (83.7%), supervisor/manager/leader support (83.3%) and reporting events (77.1%). The weakest areas related to communication about error (63.9%), hospital-level management support (60.5%) and handovers and information exchange (58.0%). Barriers to ILS use included 'it takes too long' and that many respondents must use multiple reporting systems, including mandatory hospital-level systems. These are generally not optimal for specific radiation oncology needs. Varied understanding was indicated of what and when to report. CONCLUSION: The findings report the ANZ perspective on ILS and SC, highlighting weaknesses, barriers and areas for further investigation. Differences observed in some areas suggest that a unified state, national or bi-national ILS specific to radiation oncology might eliminate multiple reporting systems and reduce reporting time. It could also provide more consistent and robust approaches to incident reporting, information sharing and analysis.

Comparison of highly pure rAAV9 vector stocks produced in suspension by PEI transfection or HSV infection reveals striking quantitative and qualitative differences.

Recent clinical successes have propelled recombinant adeno-associated virus vectors (rAAV) to the center stage for human gene therapy applications. However, the exploding demand for high titers of highly pure rAAV vectors for clinical applications and market needs remains hindered by challenges met at the manufacturing stage. The production of rAAV by transfection in suspension cells remains one of the most commonly used production platforms. In this study, we describe our optimized protocol to produce rAAV by polyethyleneimine (PEI)-mediated transfection in suspension HEK293 cells, along with a side-by-side comparison to our high-performing system using the herpes simplex virus (HSV). Further, we detail a new, robust, and highly efficient downstream purification protocol compatible with both transfection and infection-based harvests that generated rAAV9 stocks of high purity. Our in-depth comparison revealed quantitative, qualitative, and biological differences between PEI-mediated transfection and HSV infection. The HSV production system yielded to higher rAAV vector titers, higher specific yields, and a higher percentage of full capsids than transfection. Furthermore, HSV-produced stocks had a significantly lower concentration of residual host cell proteins and helper DNA impurities, but contained detectable levels of HSV DNA. Importantly, the potency of PEI-produced and HSV-produced rAAV stocks were identical. Analyses of AAV Rep and Cap expression levels and replication showed that HSV-mediated production led to a lower expression of Rep and Cap, but increased levels of AAV genome replication. Our methodology enables high-yield, high purity rAAV production and a biological framework to improve transfection quality and yields by mimicking HSV-induced biological outcomes.

Evaluating incident learning systems and safety culture in two radiation oncology departments.

INTRODUCTION: Radiation oncology patient pathways are complex. This complexity creates risk and potential for error to occur. Comprehensive safety and quality management programmes have been developed alongside the use of incident learning systems (ILSs) to mitigate risks and errors reaching patients. Robust ILSs rely on the safety culture (SC) within a department. The aim of this study was to assess perceptions and understanding of SC and ILSs in two closely linked radiation oncology departments and to use the results to consider possible quality improvement (QI) of department ILSs and SC. METHODS: A survey to assess perceptions of SC and the currently used ILSs was distributed to radiation oncologists, radiation therapists and radiation oncology medical physicists in the two departments. The responses of 95 staff were evaluated (63% of staff). The findings were used to determine any areas for improvement in SC and local ILSs. RESULTS: Differences were shown between the professional cohorts. Barriers to current ILS use were indicated by 67% of respondents. Positive SC was shown in each area assessed: 69% indicated the departments practised a no-blame culture. Barriers identified in one department prompted a QI project to develop a new reporting system and process, improve departmental learning and modify the overall ILS. CONCLUSION: An understanding of SC and attitudes to ILSs has been established and used to improve ILS reporting, feedback on incidents, departmental learning and the QA program. This can be used for future comparisons as the systems develop.

IsoTag™AAV: an innovative, scalable & non-chromatographic method for streamlined AAV manufacturing.

Demand for gene therapies capable of treating previously inaccessible targets has risen precipitously in the past decade. Adeno-associated viruses (AAVs) are the preferred vector for gene delivery because of their favorable safety profile and tissue tropism, but they have significant manufacturing challenges, with end-to-end yields as low as 10-30%. To combat these low yields, we developed IsoTag™AAV, a novel purification technology for AAV that is a departure from the chromatographic paradigm in downstream processing. This proprietary technology uses a self-scaffolding recombinant protein reagent that can improve manufacturing yields. It enables purification by cost-effective and scalable filtration processes and improves product quality with minimal optimization. Herein, we describe the development of IsoTag™AAV, provide a head-to-head comparison to industry-leading affinity chromatography (evaluation carried out through a joint research project with Capsida Biotherapeutics), and demonstrate how it can reduce cost of goods for a clinical AAV program by 25%.

The case for skin camouflage in the management of upper limb scarring - A case series.

Introduction: For some people the appearance of their hands is as important as function. Upper limb scarring can cause some patients distress. Skin camouflage is an intervention that can be used to reduce the visual impact of a scar but there is limited published evidence for its use for hand scarring. Methods: This is a case series study with a primary objective to determine whether skin camouflage reduces distress in patients with an upper limb scar and to evaluate this new service. Patients experiencing distress from an upper limb scar were recruited from a hand therapy outpatient clinic. The intervention delivered was a one hour skin camouflage session. Photographs of the upper limb pre and post skin camouflage intervention were taken. The patient-rated Michigan Hand Questionnaire (MHQ) and Derriford Appearance Scale (DAS24) were completed before treatment, at 1 week and 1 month after treatment. Results: Six participants reporting distress from an upper limb scar received skin camouflage intervention. Only three out of six participants completed all follow-up. All three showed improvement in at least two domains of the MHQ (function and aesthetics) at one month post treatment. Increased confidence during functional and work-based activities was also reported on the DAS24. Participants reported increased engagement in daily activities as a result of being able to camouflage their scars. Conclusions: This small case series shows that skin camouflage intervention may be beneficial for some patients who are experiencing distress related to an upper limb scar by increasing function and self-confidence.

Safety and Efficacy of T-DM1 Plus Neratinib in Patients With Metastatic HER2-Positive Breast Cancer: NSABP Foundation Trial FB-10.

PURPOSE: Patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer eventually develop resistance to dual-antibody therapy with trastuzumab plus pertuzumab. Mechanisms of resistance have not been well elucidated. We evaluated the safety, tolerability, and efficacy of ado-trastuzumab emtansine (T-DM1) plus neratinib in patients who progressed on trastuzumab plus pertuzumab. PATIENTS AND METHODS: In this 3 + 3 dose-escalation study, patients with metastatic breast cancer who progressed on trastuzumab, pertuzumab, and a taxane were treated with T-DM1 at 3.6 mg/kg intravenously every 3 weeks and dose-escalating neratinib at 120, 160, 200, or 240 mg/d orally. RESULTS: Twenty-seven patients were treated across four dose-levels of neratinib. Dose-limiting toxicity in cycle 1 was grade 3 diarrhea in six patients and grade 3 nausea in one; no patient experienced grade 4 diarrhea, and there were no grade 5 toxicities. Other grade 3 to 4 toxicities included nausea (11%), dehydration (11%), electrolyte abnormality (19%), thrombocytopenia (15%), elevated transaminase levels (7%), and fatigue (7%). Twelve (63%) of 19 evaluable patients had an objective response. Responses occurred at all neratinib doses. Plasma cell-free DNA at baseline showed ERBB2 (HER2) amplification in 10 of 27 patients. Deep and more durable responses occurred in patients with cell-free DNA ERBB2 amplification. Two complete responders had high expression of total HER2 and p95HER2 in baseline tissue. CONCLUSION: We report the recommended phase II dose of T-DM1 3.6 mg/kg and neratinib 160 mg/d for this combination. Possible resistance mechanisms to HER2 antibodies may be loss of the HER2 receptor and high expression of p95HER2. These data provide the basis for an ongoing phase II study to better define the activity of this regimen.

Infection and persistence of erythrovirus B19 in benign and cancerous thyroid tissues.

Human erythrovirus B19 (EVB19) is a small, pathogenic DNA virus that has been associated with a wide range of illnesses. The primary site of replication is in bone marrow-derived erythroid progenitor cells, but EVB19 DNA has been detected in a wide range of organs. Recently, studies have linked EVB19 to thyroid cancers and other thyroid diseases. Previous studies from multiple laboratories have detected EVB19 capsid proteins in Graves' disease, Hashimoto's thyroiditis, and thyroid cancer tissues. Data on viral gene expression and mechanism of infection in the thyroid are lacking. To investigate EVB19 infection and persistence in the thyroid, previously archived adult and pediatric tissue sections were examined for EVB19 DNA, RNA, and capsid proteins, as well as EVB19 receptor P-antigen and co-receptor α5ß1 integrin. EVB19 DNA and protein were detected in a majority of tissues examined (87% and 68%, respectively). Detection was similar in adult and pediatric samples. Quantification of viral genomes revealed no significant difference in the amount of viral DNA in benign, cancerous, or metastatic thyroid tissues. EVB19 capsid RNA was detected in 67% of the tissues examined, confirming at least low-level viral gene expression. Immunohistochemical staining for P-antigen and α5ß1 detected the receptor and co-receptor most frequently on normal thyroid epithelial cells. EVB19 capsid staining could be detected in tumors lacking viral receptors. These results suggest that normal thyroid epithelial cells are the initial target for EVB19 infection in the thyroid and allow for continued persistence in both normal and cancerous thyroid tissues.

Safety and efficacy of neratinib in combination with weekly paclitaxel and trastuzumab in women with metastatic HER2­positive breast cancer: an NSABP Foundation Research Program phase I study.

PURPOSE: Neratinib is an oral, small-molecule inhibitor that irreversibly binds to pan-HER (ErbB) receptor tyrosine kinases. Studies suggest that dual anti-HER therapies utilized in breast cancer patients are more efficacious than single agents in both the metastatic and neoadjuvant settings. In this phase I study, neratinib was combined with trastuzumab and paclitaxel in metastatic HER2-positive patients. METHODS: Twenty-one patients entered this dose-escalation study to determine the maximum-tolerated dose, safety, and efficacy of neratinib (120 up to 240 mg/day) with trastuzumab (4 mg/kg IV loading dose, then 2 mg/kg IV weekly), and paclitaxel (80 mg/m(2) IV days 1, 8, and 15 of a 28-day cycle) in women with HER2-positive metastatic breast cancer previously treated with anti-HER agent(s) and a taxane. RESULTS: The recommended phase II dose of neratinib with trastuzumab and paclitaxel was 200 mg/day. Common grade 3/4 adverse events were diarrhea (38 %), dehydration (14 %), electrolyte imbalance (19 %), and fatigue (19 %). With mandated primary diarrheal prophylaxis, ≥grade 3 diarrhea was not observed. Objective responses, complete (CR) and partial (PR), occurred in eight patients (38 %), with a clinical benefit of CR + PR+ stable disease (SD) ≥24 weeks in 11 patients (52 %). Median time-to-disease progression was 3.7 months. CONCLUSIONS: Dual anti-HER blockade with neratinib and trastuzumab resulted in significant clinical benefit despite prior exposure to trastuzumab, lapatinib, T-DM1, a taxane, and multiple lines of chemotherapy. In selected populations, inhibiting multiple ErbB-family receptors may be more advantageous than single-agent inhibition. Based on favorable tolerance and efficacy, this three-drug combination will be further assessed in a randomized phase II neoadjuvant trial (NSABP FB-7:NCT01008150).

Increased IL-6 detection in adult and pediatric lymphoid tissue harboring parvovirus B19.

BACKGROUND: Parvovirus B19 (B19V) is a common pathogenic virus infecting humans. Previous studies have shown evidence of B19V infection in patients with non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma using ELISA and PCR on serum specimens. B19V nonstructural protein is known to alter the expression of cellular factors including interleukin-6 (IL-6), which can increase the risk for and worsen the prognosis of lymphomas. OBJECTIVE: The objective of this study was to detect B19V capsid protein and IL-6 expression in normal and malignant lymphoid tissue, as well as any correlation between the two. STUDY DESIGN: IHCs for B19V capsid protein, IL-6, and B19V co-receptors P-antigen and α5ß1 integrin were performed on a tissue array containing 70 duplicated pediatric and adult lymphoma tissues and 5 duplicated benign lymph node sections. Cases were identified as normal, B-cell NHL, diffuse large B-cell NHL, Hodgkin's lymphoma, extranodal NK/T cell lymphoma, anaplastic large cell lymphoma, or mantle cell lymphoma. IL-6 and B19V capsid staining were quantified using a positive pixel count algorithm, and P-antigen and α5ß1 staining using a membrane quantification algorithm. RESULTS: B19V capsid protein was detected in both benign and malignant lymphoid tissue. The Spearman rank correlation coefficient analysis was performed to determine the relationship between the level of positivity for B19V and IL-6 staining, yielding an overall correlation coefficient of 0.679 (p-value<0.0001). CONCLUSIONS: Our results show a moderate correlation between the levels of positive B19V and IL-6 staining by IHC, indicating a possible role for B19V in the pathogenesis of lymphomas.

Detection of parvovirus B19 capsid proteins in testicular tissues.

OBJECTIVE: To detect B19 capsid proteins, VP1 and VP2, in testicular tissues, both normal and tumor, using immunohistochemistry. METHODS: Samples of normal, fetal, and tumor testicular tissue (n = 31) and normal testicular DNA (n = 1) were tested for the presence of B19. Immunohistochemistry staining was used for the detection of viral capsid proteins VP1 and VP2. Polymerase chain reaction with 4 primer sets was used to test for the presence of B19 DNA in a normal testicular sample. RESULTS: B19 capsid protein VP1 and VP2 was detected by immunohistochemistry in 6 (85.7%) of 7 normal testicular samples and 17 (73.9%) of 23 tumor samples. The findings from a normal fetal testicular sample were equivocal. B19 DNA was detected in normal testicular DNA with 4 of the 4 primer sets used. CONCLUSION: In contrast to previous reports, B19 capsid proteins VP1 and VP2 have now been detected in both normal and tumor testicular tissue. The persistence of B19 in a diverse range of tissues, including the testes, requires more research into the molecular mechanisms by which B19 can enter these cells, as well as the possible etiologic roles in chronic diseases, including cancer.

Parvovirus B19 infection in Hashimoto's thyroiditis, papillary thyroid carcinoma, and anaplastic thyroid carcinoma.

BACKGROUND: The human pathogenic parvovirus B19 (B19) has recently been detected in papillary thyroid carcinoma (PTC) and Hashimoto's thyroiditis (HT) tissues at a high frequency in two studies of a Chinese cohort. We wanted to extend these data to include another cohort and expand the thyroid tumor tissue types assessed. In particular, we were interested to find whether B19 also infects anaplastic thyroid carcinoma (ATC), one of the most aggressive human cancers. METHODS: Commercially available thyroid tumor tissue arrays were used to detect B19 capsid protein by immunohistochemistry in various types of thyroid tumors and disorders. The arrays were representative of the four main types of thyroid tumors, as well as other thyroid autoimmune disorders such as HT and Graves' disease, and adenomas, goiters, lymphomas, and normal thyroid tissue. In total, at least 12 different types of thyroid conditions as well as normal tissue were represented, many with multiple subjects. RESULTS: Twenty-one of the 24 (88%) PTC tumors, 3 of the 3 ATC/undifferentiated tumors, and 3 of the 3 HT tissue samples were positive for B19 capsid protein by immunohistochemistry. The localization of the protein differed based on pathological disease type, with a nuclear to cytoplasmic shift seen from unaffected to tumor tissue. CONCLUSIONS: We extend the data available on B19 detection in the thyroid to show a high correlation of virus in another cohort of PTC and HT at the protein level. We also show, for the first time, B19 infection of much more highly aggressive ATC/undifferentiated tumors. Nuclear to cytoplasmic shift in B19 protein in cancer tissue suggests a possible link between B19 and thyroid cancer pathogenesis/progression.

Brain-derived neurotrophic factor released from engineered mesenchymal stem cells attenuates glutamate- and hydrogen peroxide-mediated death of staurosporine-differentiated RGC-5 cells.

The purpose of this study was to determine the viability of cell-based delivery of brain-derived neurotrophic factor (BDNF) from genetically modified mesenchymal stem cells (MSCs) for neuroprotection of RGC-5 cells. RGC-5 cells were differentiated with the protein kinase inhibitor staurosporine (SS) and exposed to the cellular stressors glutamate or H2O2. As a neuroprotective strategy, these cells were then co-cultured across a membrane insert with mesenchymal stem cells (MSCs) engineered with a lentiviral vector for production of BDNF (BDNF-MSCs). As a positive control, recombinant human BDNF (rhBDNF) was added to stressed RGC-5 cells. After SS-differentiation RGC-5s developed neuronal-like morphologies, and a significant increase in the proportion of RGC-5s immunoreactive for TuJ-1 and Brn3a was observed. Differentiated RGC-5s also had prominent TrkB staining, demonstrating expression of the high-affinity BDNF receptor. Treatment of SS-differentiated RGC-5s with glutamate or H2O2, produced significant cell death (56.0 +/- 7.02 and 48.90 +/- 4.58% of control cells, respectively) compared to carrier-solution treated cells. BDNF-delivery from MSCs preserved more RGC-5 cells after treatment with glutamate (80.0 +/- 5.40% cells remaining) than control GFP expressing MSCs (GFP-MSCs, 57.29 +/- 1.89%, p < 0.01). BDNF-MSCs also protected more RGC-5s after treatment with H2O2 (65.6 +/- 3.47%) than GFP-MSCs (46.0 +/- 4.20%, p < 0.01). We have shown survival of differentiated RGC-5s is reduced by the cellular stressors glutamate and H2O2. Additionally, our results demonstrate that genetically modified BDNF-producing MSCs can enhance survival of stressed RGC-5 cells and therefore, may be effective vehicles to deliver BDNF to retinal ganglion cells affected by disease.