Persisting Cancer in Black Salve Treated Skin Lesions: Results of a Large 5 Year Retrospective Analysis of Australian Histopathology Specimens.

BACKGROUND: To date there has been minimal research on the use of black salve escharotics. Whether cancer persistence is a frequent finding in treatment areas, the types of lesion being treated by patients, whether rural patients are more likely to use black salve and whether current government prevention initiatives are succeeding are all issues needing investigation. METHODS: This study was a large national retrospective black salve pathology case series from 2015 to 2019. Five private pathology companies with 1471 collection centers located in 5 of the 8 Australian states and Territories provided de-identified skin pathology report information where black salve treatment had been documented in the pathology request. RESULTS: Over the 5-year period 409 patients had treated 475 lesions with black salve. Benign lesions were present at the treatment site in 18% of cases; persisting cancer was found in 34.2% of the remaining black salve treated areas. The majority of treatment areas were located on the head and neck. Black salve caused necrosis of normal tissue when treating benign lesions, refuting claims of cancer specificity. Likelihood of black salve use increased with rurality based on Modified Monash (MM) scores. Black salve use, despite regulatory efforts, appears to be increasing with specimen numbers more than doubling from 2015 to 2019. CONCLUSIONS: Patients undergoing histopathological assessment of black salve treatment areas have high rates of cancer persistence. Patients are applying black salve to benign lesions and lesions in cosmetically sensitive areas. Rural patients have higher proportional rates of black salve use. The increasing incidence of black salve pathology specimens suggests current Australian black salve public health initiatives are failing.

Assessment of patient-to-patient and intra-individual human abdominal skin immune cell variability.

Introduction: The objective of the study was to characterize the baseline intra-individual and inter-individual variability of immune cell subsets within abdominoplasty skin specimens. Methods: Abdominoplasty biopsies were taken from 5 patients and analysed using the Vectra 3 automated quantitative pathology imaging system with inForm software. Results: Adjacent skin regions demonstrated intra-patient variability in immune subset counts ranging from 1- to 5-fold. Inter-variability between patients was approximately 2- to 7-fold for most subsets, except for HLA-DR+ antigen presenting cells, which varied 19-fold. Conclusions: Our data highlight the importance of including multiple patients and multiple patient samples when designing dermatological studies that utilise abdominoplasty skin.

Understanding of black salve toxicity by multi-compound cytotoxicity assays.

BACKGROUND: Black salve is a controversial complementary and alternative medicine (CAM) associated with skin toxicity and skin cancer treatment failures. Black salve formulations vary between manufacturers and contain a number of botanical and synthetic constituents. The skin cancer cytotoxicity of a number of these constituents has not been assessed to date. The alkaloids from the rhizomes of Sanguinaria canadensis, a key black salve ingredient, have had their single compound cytotoxicity assessed; however, whether they possess synergistic cytotoxicity with other compounds has not been studied and is of direct clinical relevance. This research aimed to improve our understanding of the skin cancer cytotoxicity of black salve constituents. METHODS: The cytotoxicity of individual and combination black salve constituents were assessed against the A375 melanoma and A431 squamous cell carcinoma cell lines. Cytotoxicity was determined using the Resazurin assay with fluorescence measured using a Tecan Infinite 200 Pro Microplate reader, compound cytotoxicity being compared to that of the topical cancer therapeutic agent, 5- fluouracil. Docetaxal was used as a positive control. Dunnetts p value was used to determine whether significant synergistic cytotoxicity was present. RESULTS: Sanguinarine was the most cytotoxic compound tested with a 24-hour IC50 of 2.1 µM against the A375 Melanoma cell line and 3.14 µM against the A431 SCC cell line. All black salve constituents showed greater cytotoxicity against the two skin cancer cell lines tested than the skin cancer therapeutic 5-Fluouracil with 24 hours of compound exposure. Chelerythrine and minor Quaternary Benzophenanthridine Alkaloids (QBAs) present in black salve, at concentrations not having a cytotoxic effect by themselves, boosted the cytotoxic effects of sanguinarine. This could be a synergistic rather than additive cytotoxic effect although the synergistic effect was cell line and concentration dependent. CONCLUSIONS: Black salve contains several cytotoxic compounds, a number of which have been found to possess synergistic cytotoxicity for the first time against skin cancer cell lines. In addition, these compounds together increase the overall cytotoxic effect. Assessing multi-compound cytotoxicity in herbal medicine can provide additional information about both their therapeutic and toxicity potential. As black salve is currently being used by patients, further cytotoxicity work should be undertaken to assess whether synergistic cytotoxicity exists when tested in normal skin cells.

Comparison of skin biopsy sample processing and storage methods on high dimensional immune gene expression using the Nanostring nCounter system.

BACKGROUND: Digital multiplex gene expression profiling is overcoming the limitations of many tissue-processing and RNA extraction techniques for the reproducible and quantitative molecular classification of disease. We assessed the effect of different skin biopsy collection/storage conditions on mRNA quality and quantity and the NanoString nCounter™ System's ability to reproducibly quantify the expression of 730 immune genes from skin biopsies. METHODS: Healthy human skin punch biopsies (n = 6) obtained from skin sections from four patients undergoing routine abdominoplasty were subject to one of several collection/storage protocols, including: i) snap freezing in liquid nitrogen and transportation on dry ice; ii) RNAlater (ThermoFisher) for 24 h at room temperature then stored at - 80 °C; iii) formalin fixation with further processing for FFPE blocks; iv) DNA/RNA shield (Zymo) stored and shipped at room temperature; v) placed in TRIzol then stored at - 80 °C; vi) saline without RNAse for 24 h at room temperature then stored at - 80 °C. RNA yield and integrity was assessed following extraction via NanoDrop, QuantiFluor with RNA specific dye and a Bioanalyser (LabChip24, PerkinElmer). Immune gene expression was analysed using the NanoString Pancancer Immune Profiling Panel containing 730 genes. RESULTS: Except for saline, all protocols yielded total RNA in quantities/qualities that could be analysed by NanoString nCounter technology, although the quality of the extracted RNA varied widely. Mean RNA integrity was highest from samples that were placed in RNALater (RQS 8.2 ± 1.15), with integrity lowest from the saline stored sample (RQS < 2). There was a high degree of reproducibility in the expression of immune genes between all samples with the exception of saline, with the number of detected genes at counts < 100, between 100 and 1000 and > 10,000 similar across extraction protocols. CONCLUSIONS: A variety of processing methods can be used for digital immune gene expression profiling in mRNA extracted from skin that are comparable to snap frozen skin specimens, providing skin cancer clinicians greater opportunity to supply skin specimens to tissue banks. NanoString nCounter technology can determine gene expression in skin biopsy specimens with a high degree of sensitivity despite lower RNA yields and processing methods that may generate poorer quality RNA. The increased sensitivity of digital gene expression profiling continues to expand molecular pathology profiling of disease.

Assessing the risk of epidemic dropsy from black salve use.

Epidemic dropsy is a potentially life-threatening condition resulting from the ingestion of argemone oil derived from the seeds of Argemone mexicana Linn. Exposure to argemone oil is usually inadvertent, arising from mustard cooking oil adulteration. Sanguinarine, an alkaloid present in argemone oil, has been postulated as a causative agent with the severity of epidemic dropsy correlating with plasma sanguinarine levels. Cases of epidemic dropsy have also been reported following the topical application of argemone containing massage oil. Black salve, a topical skin cancer therapy also contains sanguinarine, but at significantly higher concentrations than that reported for contaminated massage oil. Although not reported to date, a theoretical risk therefore exists of black salve inducing epidemic dropsy. This literature review explores the presentation and pathophysiology of epidemic dropsy and assesses the risk of it being induced by black salve.

Black salve in a nutshell

Background: Black salve is an alternative therapy increasingly chosen by patients to selfmanage their skin lesions. It is promoted as an effective, safe and natural skin cancer treatment, but such claims are not evidence-based, and serious complications have been reported. The sale of black salve in Australia is illegal. Objective: The aim of this article is to educate general practitioners (GPs) about black salve, enabling informed discussion with patients considering using black salve. An overview of the scientific literature is presented. Discussion: Case reports have described significant morbidity and even mortality associated with the use of black salve. Despite this, black salve is readily accessible to the public online; a simple internet search yields multiple links to websites endorsing black salve as an effective natural skin cancer remedy. As GPs are often called on in the initial presentation of skin complaints, they are well positioned to ask patients about their use of black salve and educate them about its risks.

Carcinogenic potential of sanguinarine, a phytochemical used in 'therapeutic' black salve and mouthwash.

Black salves are escharotic skin cancer therapies in clinical use since the mid 19th century. Sanguinaria canadensis, a major ingredient of black salve formulations, contains a number of bioactive phytochemicals including the alkaloid sanguinarine. Despite its prolonged history of clinical use, conflicting experimental results have prevented the carcinogenic potential of sanguinarine from being definitively determined. Sanguinarine has a molecular structure similar to known polyaromatic hydrocarbon carcinogens and is a DNA intercalator. Sanguinarine also generates oxidative and endoplasmic reticulum stress resulting in the unfolded protein response and the formation of 8-hydroxyguanine genetic lesions. Sanguinarine has been the subject of contradictory in vitro and in vivo genotoxicity and murine carcinogenesis test results that have delayed its carcinogenic classification. Despite this, epidemiological studies have linked mouthwash that contains sanguinarine with the development of oral leukoplakia. Sanguinarine is also proposed as an aetiological agent in gallbladder carcinoma. This literature review investigates the carcinogenic potential of sanguinarine. Reasons for contradictory genotoxicity and carcinogenesis results are explored, knowledge gaps identified and a strategy for determining the carcinogenic potential of sanguinarine especialy relating to black salve are discussed. As patients continue to apply black salve, especially to skin regions suffering from field cancerization and skin malignancies, an understanding of the genotoxic and carcinogenic potential of sanguinarine is of urgent clinical relevance.

A Review of Black Salve: Cancer Specificity, Cure, and Cosmesis.

Black salve is a topical escharotic used for the treatment of skin cancer. Although promoted as a safe and effective alternative to conventional management by its proponents, limited clinical research has been undertaken to assess its efficacy and potential toxicities. Patients are increasingly utilizing the Internet as a source of health information. As a minimally regulated space, the quality and accuracy of this information vary considerably. This review explores four health claims made by black salve vendors, investigating its natural therapy credentials, tumour specificity, and equivalence to orthodox medicine in relation to skin cancer cure rates and cosmesis. Based upon an analysis of in vitro constituent cytotoxicity, in vivo post black salve histology, and experience with Mohs paste, black salve is likely to possess normal tissue toxicity with some cancer cell lines being relatively resistant to its effects. This may explain the incongruous case study reports of excessive scarring, deformity, and treatment failure.

Sanguinaria canadensis: Traditional Medicine, Phytochemical Composition, Biological Activities and Current Uses.

Sanguinaria canadensis, also known as bloodroot, is a traditional medicine used by Native Americans to treat a diverse range of clinical conditions. The plants rhizome contains several alkaloids that individually target multiple molecular processes. These bioactive compounds, mechanistically correlate with the plant's history of ethnobotanical use. Despite their identification over 50 years ago, the alkaloids of S. canadensis have not been developed into successful therapeutic agents. Instead, they have been associated with clinical toxicities ranging from mouthwash induced leukoplakia to cancer salve necrosis and treatment failure. This review explores the historical use of S. canadensis, the molecular actions of the benzophenanthridine and protopin alkaloids it contains, and explores natural alkaloid variation as a possible rationale for the inconsistent efficacy and toxicities encountered by S. canadensis therapies. Current veterinary and medicinal uses of the plant are studied with an assessment of obstacles to the pharmaceutical development of S. canadensis alkaloid based therapeutics.