TRUPCR DPYD Testing
In the current age of medical development, the fight against cancer has never been more prevalent. The disease was alone responsible for almost ten million deaths, approximately 1 in 6 deaths, in 2020 according to the World Health Organization (WHO) making it the leading cause of death worldwide currently.
Today, we understand that cancer arises from typically multi-faceted DNA mutations or modifications within healthy cells turning them into cancerous cells. Typically, these mutations result in the cells exhibiting one or more of what are known as a “Hallmark of Cancer”, a term coined in 2000 by Douglas Hanahan and Robert Weinberg (1). Originally, six of these biological abilities were described, but the list has been expanded to eight capabilities and two enabling capabilities in 2011. These are as follows:
- Resisting Apoptosis
Apoptosis is a type of programmed cell death which comes into action when they become damaged or abnormal. However, in the case of cancerous cells, the mechanisms in which this is facilitated are altered, making the cell unable to detect changes which would otherwise trigger apoptosis, effectively preventing the abnormal cell from dying.
- Replicative Immortality
Non-cancerous cells, after a certain number of cycles of mitosis, will either enter senescence or undergo apoptosis. This limit is known as the Hayflick Limit (2) and protects the organism overall from mutated cells that may become cancerous. It is measured by sections of DNA at the ends of each chromosome known as the “telomeres”, which are maintained by the telomerase enzyme but are also shortened after each mitosis cycle. Besides cancerous cells being able to evade cell death, as previously discussed, they can also alter the activity of telomerase, effectively keeping the telomeres intact. In turn, this allows the cells to replicate regardless of the number of cell divisions or genetic status of the cell.
- Inducing Angiogenesis
Cancer cells require far more resources than a non-cancerous cell would, namely oxygen. This is to allow for the increase in cellular respiration to produce the Adenosine Triphosphate (ATP) required for their high demands stemming from e.g., their increased replication capacity. To achieve this, some mutations in cancerous cells promote the production of proteins which encourage the generation of blood vessels to the tumor, thereby increasing access to oxygen.
- Tissue Invasion & Metastasis
The ability to outgrow their local environment and migrate around the patient through other tissues and/or through blood/lymphatic vessels.
- Self-Sufficiency in Growth Signals
With this ability, cancer cells do not require the stimulation of external factors to multiply. They circumvent this requirement via producing these signals themselves in what is known as autocrine signaling, or by manipulating the cell pathways by either permanently activating them, or by inactivating their negative feedback pathways, which act as a stopper, resulting in uncontrolled cell growth.
- Insensitivity to Anti-Growth Signals
Normally, tumor suppressor genes are responsible for gatekeeping the growth and division of cells. They do this by taking information from the cell to ensure that it is ready to divide and stop it from doing so, if it isn’t. The other method is by contact inhibition, whereby, a population cells which completely fill a space, sense this and halt their growth. However, in cancerous cells, these methods are either inhibited, or altered so that they do not carry out their function, allowing them to grow irrespective of whether they need to or not.
- Deregulated Metabolism
Most cancer cells use alternate metabolic pathways as opposed to standard aerobic respiration. This is known as the Warburg effect. Cells exhibiting the Warburg effect prefer glycolysis and lactic acid fermentation which occurs in the cytosol, preventing mitochondria carrying out its normal function. Due to less ATP being produced because of less efficient methods being used; the deactivation of the mitochondria can occur.
- Evading the Immune System
Even though cancer cells are known to increase inflammation and angiogenesis, they are known to avoid interactions with the body’s immune system. This is done by the production of “don’t kill me” proteins or by removal of proteins that promote the immune response.
- Genome Instability
Due to the increased number of cell divisions that cancerous cells undergo, there is an inherent increased likelihood of genetic mutations occurring. This is an enabling factor due to the biological capabilities of the hallmarks of cancer being made possible due to genomic changes in normal, healthy cells.
Inflammation causes the breakdown of the extracellular matrix. This in turn, provides an easier pathway for the metastasis of cancerous cells, and provides space for angiogenesis to occur, providing any tumor with the space and resources for their proliferation.
Overcoming the Hallmarks of Cancer:
Treatments for cancer typically work by counteracting one or more of these hallmarks of cancer. One such treatment is fluorouracil (5-FU). This treatment is a type of antimetabolite chemotherapy as the structure of the molecule closely resembles naturally occurring molecules within cells. This allows it to insert itself into cells, interfering with their growth and/or division. In this case, 5-FU is similar to deoxyuridine monophosphate (dUMP), the substrate for enzymes responsible for DNA replication, namely thymidylate synthase. Ordinarily, thymidylate synthase is responsible for the synthesis of thymidylate monophosphate (dTMP), one of the nucleotides found in DNA. However, with the introduction of 5-FU acting as a competitor of dUMP for thymidylate synthase activity, dUMP is not converted to dTMP. Consequently, the amount of dTMP within cells becomes limited or non-existent, leading to cells to undergo cell death by thymine deprivation.
As a result of its function, 5-FU is a cytotoxic molecule results in cell death depending on its concentration. However, cells produce the enzyme dihydropyrimidine dehydrogenase (DPD), which is responsible for the metabolism of pyrimidines such as 5-FU. DPD allows for the control of 5-FU within healthy cells to prevent them from undergoing thymineless death themselves by effectively reducing its cellular concentration below critical levels.
However, issues arise when a patient has DPD deficiency which is caused by autosomal recessive mutations on the DPYD gene. There are four mutations of note: c.1905+1G>A, c.1679T>G, c.2846A>T and c.1129–5923C>G. It has been found that as many as 8% of people have at least a partial deficiency of DPD because of any one of these mutations.
Symptoms of this deficiency does not typically result in symptoms unless the deficiency is complete or severe, however, a cancer patient undergoing treatment using 5-FU would experience more dangerous side-effects than an individual without the mutation. This is because the drug would not be metabolized and instead build up within the body. These side effects include:
- Severe diarrhea.
- Sickness and vomiting which may lead to dehydration.
- Decreased blood cell levels, increasing infection risk, bleeding and breathlessness.
To prevent this level of toxicity in these individuals, medical professionals have begun ordering and carrying out a blood test on would be patients to see if they carry any of the mutations responsible for DPD deficiency. One such test is conducted via qPCR.
At TRUPCR® Europe, we produce the DPYD Mutations Detection qPCR kit. The kit uses ARMS PCR to detect and differentiate between the four DPYD mutations (c.1905+1G>A, c.1679T>G, c.2846A>T and c.1129–5923C>G) known to cause DPD deficiency, allowing for screening to results in just a short, 40-minute PCR run.