Hemolysis is the most prevalent pre-analytical interfering factor and a major source of error in laboratory analysis. The examination of samples post-centrifugation can provide valuable information regarding pre-analytical interferences. In this unusual case, a patient\'s plasma specimen was cherry-red after centrifugation, which is most usually indicative of hemolysis. However, subsequent investigations ruled out common hemolysis causes. We eventually determined that the patient\'s cherry-red plasma was most likely caused by other factors in the patient\'s medical history, including cancer treatment with PV-10 (rose bengal disodium 10%). We then conducted an interference study to comprehensively assess the effects of PV-10 on various biochemical tests, especially liver function tests and bilirubin levels. The findings indicate that PV-10 has varying effects on different biochemical assays and test results should be examined individually. This report underlines the need for awareness of potential drug interference on laboratory tests for better result interpretation and making clinical decisions.

PV-10 is a 10% formulation of rose bengal sodium that has potent immunotherapeutic and anti-cancer activity against various tumors, including metastatic melanoma and refractory neuroblastoma. Currently, PV-10 is undergoing clinical testing for refractory metastatic neuroendocrine cancer and melanomas. However, preclinical investigation of PV-10 activity and its mechanisms against phenotypically and molecularly diverse adult solid tumors had not been conducted. In a panel of human cell lines derived from breast, colorectal, head and neck, and testicular cancers, we demonstrated that PV-10 induces cytotoxicity by apoptotic and autophagic pathways involving caspase-mediated PARP cleavage, downregulation of SQSTM1/p62, and upregulation of beclin-1. Treatment with PV-10 also consistently reduced phosphorylation of WNK1, which has been implicated in cancer cell migration and autophagy inhibition. By wound healing assay, PV-10 treatment inhibited the migration of cancer cells. Finally, significant inhibition of tumor growth was also noted in tumor-bearing mice treated with PV-10 by intralesional or systemic administration. In addition to known PV-10-mediated tumor-specific cytotoxic effects, we identified the mechanisms of PV-10 and provide new insights into its effect on autophagy and metastasis. Our data provide essential mechanism-based evidence and biomarkers of activity to formulate clinical studies of PV-10 in the future.

Although immune checkpoint inhibitors have become the standard of care for many tumours, the majority of patients fail to achieve sustained benefit, often owing to the lack of a T-cell inflamed tumour microenvironment (TME). Directly injected intratumoural therapies present a potential strategy to induce T-cell inflammation and convert a \'cold\' immune-inert TME into a \'hot\' immune-inflamed TME. Various approaches including chemoablation, oncolytic viral therapy, cytokines and agents targeting innate immunity such as Toll-like receptor agonists and stimulator of interferon genes agonists are in clinical development. Thus far, melanoma has led the way in intratumoural drug development owing to its relative immunogenicity and propensity for cutaneous metastasis easily amenable to injections. However, intratumoural therapies are moving to other tumour types and advances in endoscopic and interventional radiological techniques are allowing these agents to be injected into visceral lesions. This review provides an overview of the current status of intratumoural therapies in oncology, as well as future directions regarding therapeutic niches and appropriate trial design for intratumoural agents.

UNASSIGNED: Neuroblastoma is the most common extracranial cancer in children. Although the prognosis for low-risk neuroblastoma patients is good, the 5-year survival rates for high-risk and relapsed patients are low. The poor survival rates for these patients demonstrate the need for novel therapeutic approaches to treat this disease. PV-10 is a sterile 10% solution of Rose Bengal that has previously been shown to induce cell death in a range of adult cancers, providing the rationale for studying the activity of PV-10 against neuroblastoma in preclinical studies.
UNASSIGNED: The effects of PV-10 on neuroblastoma were investigated in vitro. Cytotoxicity assays were performed using the alamar blue assay on the following cell lines: SK-N-AS, SK-N-BE(2), IMR5, LAN1, SHEP, and SK-N-SH neuroblastoma cells, SK-N-MC neuroepithelioma cells, and normal primary, BJ, and WI38 fibroblasts. Phase-contrast, fluorescence, and time-lapse video microscopy; flow cytometry; and Western blotting were used to investigate the effects of PV-10 on SK-N-AS and IMR5 cells. Synergy with commonly used anticancer drugs was determined by calculation of combination indices in SK-N-AS and IMR5 cells. Mouse xenograft models of SK-N-AS and IMR5 tumors were also used to evaluate the efficacy of PV-10 in vivo.
UNASSIGNED: In vitro preclinical data demonstrate that pharmacologically relevant concentrations of PV-10 are cytotoxic to neuroblastoma cell lines. Studies to investigate target modulation in neuroblastoma cell lines show that PV-10 disrupts lysosomes, decreases the percentage of cells in S phase, and induces apoptosis in a concentration-, time-, and cell-line-dependent manner, and we also identify agents that are synergistic with PV-10. Furthermore, experiments in xenograft mouse models show that PV-10 induces tumor regression in vivo.
UNASSIGNED: Our study provides preclinical data on the efficacy of PV-10 against neuroblastoma and provides rationale for the development of an early phase clinical trial of this agent in relapsed and refractory neuroblastoma patients.

OBJECTIVE: Isolated limb infusion (ILI) and intralesional PV-10 are well described locoregional therapies for in-transit melanoma. The objective of this study was to assess the effect of these treatments on survival outcomes within a cohort matched for key characteristics.
METHODS: Patients were treated using ILI or intralesional PV-10 at a single institution and the data prospectively recorded. Propensity score matching was performed using key covariates within a logistic regression model. The primary outcome was the melanoma-specific survival.
RESULTS: Seventy-two patients nonrandomized were successfully matched. Both treatments produced similar best overall responses. The median melanoma-specific survival (MSS) was 74.4 months from ILI and 36.4 months from PV-10 treatments (P = 0.164). Within the ILI subgroup, the 12-, 24-, 36- and 60-month MSS rates were 85.3%, 75.3%, 60.1%, and 60.1%, respectively. From the time of PV-10 the corresponding 12-, 24-, 36-, and 60-month MSS rates were 82.6%, 70.0%, 53.9%, and 35.9%. On multivariate analysis, there was a significant difference in survival comparing completely with noncomplete responders ( P = 0.031).
CONCLUSIONS: These findings demonstrate that ILI and PV-10 treatments for in-transit disease produce comparable long-term survival. Both therapies have reproducible response rates and predominantly localized and tolerable side-effects.

Advances in treatment of melanoma with systemic immunotherapies continue, with promising findings for anti-PD-1 agents combined with ipilimumab. Still, an unmet need persists because of populations ineligible for systemic immunotherapies, incomplete cure/response rates, toxicities and extreme costs. Also, potential for effective use of intralesional therapies remains, especially for local regional disease, but also for benefits of local ablation and adjuvant systemic host tumor-specific responses. Clinical trials of T-VEC, PV-10, CAVATAK and electroporation with plasmid IL-12 have demonstrated favorable, durable responses. Initial experience combining T-VEC, the agent furthest along in testing, with ipilimumab revealed higher complete and overall response rates than with either agent alone. Intralesional therapies may offer a treatment tool in the growing therapeutic armamentarium against this lethal disease.

BACKGROUND: Patients with in-transit melanoma metastases frequently experience high rates of recurrence, limited overall survival and reduced quality of life. After promising results within a Phase II, multi-center study, PV-10 treatment was continued at our institution for patients with in-transit disease.
METHODS: An open-label, non-randomized, prospective study was performed at the Princess Alexandra Hospital, Queensland, Australia. Patients were treated with PV-10 in accordance with the treatment protocol established during a previous Phase II study. The primary outcome was the complete response of treated lesions.
RESULTS: Forty-five patients were enrolled over a total of 82 treatment episodes from July 2008 to December 2015. With sequential PV-10 treatments the complete response rate was 42% and overall response rate 87% on an intention to treat analysis. The median follow-up duration was 22 months and the median overall survival was 25 months from first PV-10 treatment. Having fewer than 15 metastases at the time of treatment was associated with a complete response (P = 0.03).
CONCLUSIONS: Intralesional PV-10 provided rapid lesion-specific ablation of melanoma metastases with well-tolerated local effects and minimal systemic adverse events. This therapy should be considered for patients with multiple accessible deposits within the spectrum of low to moderate disease volume.

The emergence of novel intralesional therapies have dramatically changed the treatment landscape for melanoma. The heterogeneous presentation of melanoma continues to pose challenges for clinicians, especially when dealing with advanced locoregional disease. Intralesional therapies have the benefit of causing local tumor destruction, while minimizing systemic toxicity. Moreover, the integration of immunotherapeutic agents into intralesional compounds has resulted in the additional benefit of a bystander effect, whereby untreated distant lesions also derive a benefit from treatment. Intralesional therapy has assumed an important role in the management of unresectable, locoregional disease for melanoma. Areas covered: Multiple intralesional agents have been studied over the years, with only a few demonstrating promising results. This review will provide an overview of the different intralesional agents for melanoma. Mechanisms of action, clinical efficacy, and side effects will be the primary focus. Expert commentary: Treatment options for advanced melanoma continue to evolve. Attractive new therapies delivered by an intralesional route has demonstrated promising results, with minimal side effects. The ideal treatment strategy for melanoma will remain a multimodal approach; intralesional therapy provides an additional tool in the treatment armamentarium for melanoma.

BACKGROUND: In-transit and recurrent dermal or subcutaneous melanoma metastases represent a significant burden of advanced disease. Intralesional Rose Bengal can elicit tumor selective ablation and a T-cell mediated abscopal effect in untreated lesions. A subset of patients in a phase II trial setting received external beam radiotherapy to their recurrent lesions with complete or partial response and no significant acute radiation reaction.
METHODS: An open-label, single-arm phase II study was performed to assess the efficacy and safety of PV-10 followed by hypofractionated radiotherapy. Patients had in-transit melanoma metastases suitable for IL therapy and radiotherapy.
RESULTS: Fifteen patients were enrolled and thirteen completed both treatment components. The overall response rate was 86.6% and the clinical benefit was 93.3% on an intention to treat analysis (CR 33.3%, PR 53.3%, SD 6.7%). The median follow up duration was 19.25 months. Size of metastases (<10 mm) predicted lesion complete response (74.6%). Treatment was well tolerated with no associated grade 4 or 5 adverse events.
CONCLUSIONS: The combination of PV-10 and radiotherapy resulted in lesion-specific, normal tissue-sparing, ablation of disease with minimal local or systemic adverse effects.

OBJECTIVE: Patients with in-transit melanoma metastasis have longer median survival than patients with distant metastatic disease. Furthermore, local disease control is an important endpoint for symptom management. The treatment of unresectable loco-regional recurrence or in-transit disease has been historically managed with a combination of treatments including surgery, radiotherapy, isolated limb infusion or perfusion as well as systemic therapies. Intralesional PV-10 has been used at Peter MacCallum Cancer Centre since 2010, and the current report presents a retrospective analysis of patient outcomes, reporting the response rates, durability of responses, and observed toxicities.
METHODS: Records were analyzed retrieving details of 19 patients treated with PV-10 over a 4-year period from 2010 to 2014. Medical records were reviewed for these patients and data extracted.
RESULTS: Nineteen patients with in-transit melanoma were treated with intralesional PV-10 between 2010 and 2014. Disease control (complete or partial response or disease stability) was achieved in 68% of patients with 26% having a complete response. This was achieved with minimal associated toxicity.
CONCLUSIONS: PV-10 is an effective, durable, well-tolerated treatment tool with an acceptable side effect profile for the management of unresectable in-transit melanoma. J. Surg. Oncol. 2016;114:380-384. © 2016 Wiley Periodicals, Inc.