A molecular multiplex ‘rapid test’ for liquid biopsy applications

Early diagnosis of cancer is a major public health concern. Liquid biopsy offers a non-invasive, easy, quick and convenient alternative approach for early diagnosis of cancer and real-time monitoring of tumor progression. Liquid biopsies have the potential to assess various biomarkers in several body fluids for early detection of cancer and to provide guidance treatment to select and monitor the appropriate therapy. It is mainly represented by blood circulating tumor cells, circulating tumor DNA (ctDNA), circulating microRNAs and exosomes that are secreted by the tumors. Using body fluid samples, liquid biopsies may be the ideal approach for the detection of these biomarkers and they are expected to provide more valid information than the invasive tissue biopsy. Also, the ease of monitoring the disease can enable clinicians to adopt the optimal therapeutic strategy.

Schematic illustration of the multiplex rapid test for the detection of the normal cell-free DNA and three single-point mutations of the KRAS gene (left) and the results of the test of blood samples form a healthy individual and a patient with colorectal cancer (right). In the healthy sample, a red spot is formed, due to accumulation of gold nanoparticles, only for the normal gene, while for the patient’s sample, three red spots are formed, one for the normal gene that is always present in blood circulation, and two for mutations 1 and 3.

The presence of circulating tumor DNA (ctDNA) or cell-free DNA (cfDNA), derived from tumors in blood circulation, is of major importance for early cancer detection, monitoring tumor progress or diagnosing the presence of activating mutations to guide treatment. Non-invasive molecular analysis of circulating tumor DNA is a promising application in personalized cancer management. The discrimination of ctDNA from normal cfDNA includes the detection of specific mutations located at the tumor DNA. These mutations are present only in the genomes of cancer cells and are not present in the DNA of normal cells of the same individual. The presence of these tumor-specific molecular alterations assures that ctDNA is an exquisite biologic specificity as a potential biomarker. The ctDNA that carries the mutations, often represents only a small fraction (<1.0%) of total cfDNA. Therefore, standard sequencing methods can detect mutated ctDNA fragments only in patients with advanced tumor stage. In addition, the half-life of cfDNA into blood circulation ranges from 15 min to 2.5 h, which enables cfDNA analysis to be considered as a real-time monitoring of the disease.

A new molecular and multiplex ‘rapid test’ was developed, for the first time, for the detection of ctDNA in blood samples for liquid biopsy applications. The detection is visual by naked eye based on gold nanoparticles used as reporters. Three major single-point DNA mutations of the KRAS gene that are related to colorectal and other types of cancer, along with the normal cell-free gene, are simultaneously detected by a single test in blood samples of healthy individuals and patients. The advantages of this rapid test are the simplicity, cost-effectiveness, rapid analysis, high detectability and specificity, while no instrumentation is needed for the detection. The test is also universal, meaning that it can be applied for the detection of any other ctDNA molecules and single-point mutations, because the molecular recognition takes place prior to the analysis with the strip test.

Despina Kalogianni
University of Patras, Greece

 

Safety assessment of Cannabidiol (CBD) as novel food

EFSA identifies scientific gaps and uncertainties

Cannabidiol, commonly referred to as CBD, is a substance either extracted from Cannabis sativa L. plants or synthesised chemically. At present, the only authorised CBD product on the European Union (EU) market is Epidyolex^®, a drug used as adjunctive therapy for seizures associated with specific forms of childhood epilepsy or tuberous sclerosis complex.

In November 2020, the Court of Justice of the EU concluded that CBD should not be considered a narcotic drug and, as such, could qualify as food.  Since human consumption of CBD prior to 15 May 1997 was not demonstrated, the European Commission considers CBD to qualify as a novel food under EU legislation. These decisions generated an unprecedented wave of applications for the use of CBD in foodstuffs and food supplements: as of mid-March 2022, the European Commission has received more than 150 dossiers, 19 of which have so far been considered suitable for risk assessment by EFSA under the Novel Food Regulation (EU) 2015/2283.

EFSA mandated its Panel on Nutrition, Novel Foods and Food Allergens (NDA) to provide an overview of the available literature data on the safety of CBD as a food ingredient and supplement. In practice, the Panel analysed information available from both animal and human studies investigating the effects of oral consumption of CBD.

This led to the publication of a statement on 7 June, 2022, highlighting several areas where data are either missing or inadequate for the safety assessment of CBD as novel food. For example, available human studies were oftentimes carried out in patients treated with other medications, at high therapeutic doses, and/or designed to determine the efficacy of Epidyolex^®. But while adverse effects are tolerated for drugs for which remedial benefits outweigh the risks, a food must be demonstrated to be safe in the healthy general population under the uses and use levels proposed by the applicants.

In the statement, the NDA Panel describes gaps and uncertainties regarding the effects of CBD consumption on the liver, gastrointestinal tract, endocrine and nervous systems, as well as one’s psychological function. Animal studies also show adverse effects on reproductive and developmental functions that require further investigation.

The NDA Panel concluded that the safety of CBD cannot be established based on the currently publicly available scientific data. It is now the responsibility of the applicants seeking authorisation for CBD novel foods intended as food ingredients and/or food supplements to clarify these uncertainties and fill these data gaps.

Océane Albert, Andrea Germini, Eirini Kouloura, Fernando Rivero Pino,
Annamaria Rossi, Ermolaos Ververis
EFSA CBD Task Force