Each cell contains a detailed blueprint of the genetic information in the form of a DNA double strand (quasi the book of life). The DNA in turn is composed of 4 different building blocks, the nucleotide bases (A, T, G and C) in variable order. This building plan (genetic code), which is specific to every human being, is needed to be able to produce, among other things, proteins, which every cell needs to function.
Changes (mutations) in the DNA can therefore lead to a change at the protein level. It is known that mutations and resulting changes in the cell or the entire organism can lead to certain diseases such as cancer.
By identifying the underlying mutations of a cancer disease, specific cancer therapies can be identified for individual patients (personalised medicine). One possibility for this is the PCDx test. This diagnostic tool combines sequencing (next generation sequencing) with immunohistochemistry. Next Generation Sequencing is a method to determine the exact sequence of bases in the DNA. This allows deviations from normal DNA (mutations) to be detected. Based on the changes found at DNA and protein level, conclusions can be drawn about the effectiveness of drugs.
Next Generation Sequencing - Basic Priciple
Next Generation Sequencing (NGS) is an umbrella term for a class of methods that can decode the sequence of nucleotides in DNA more quickly than older methods. NGS is based on a cellular mechanism that duplicates cellular DNA. In the body, cells must divide constantly in order to be able to produce new ones. To do this, the DNA must also be doubled. Doubling is achieved by splitting the double strand. The opposite bases of the two strands in the double strand are complementary. This means that if you know the base sequence of one strand, you also know the base sequence of the other strand. Therefore, one strand can serve as a template to synthesize a double strand again.
This process of DNA amplification can also be simulated in the test tube. The Next Generation Sequencing methods use this procedure and detect during the process exactly which of the 4 possible bases has been incorporated. Different companies offer different methods, all of them insisting on the detection of base insertions in a high-throughput process. For speed, short DNA fragments must be able to be massively sequenced in parallel. Different systems are available for this purpose. A frequently used sequencing method comes from the company Illumina.
Illumina sequencing is a popular type of next generation sequencing methods. Here, the incorporation of the different bases is measured via fluorescence signals. In order to use this system as a high-throughput method, the DNA to be sequenced must be broken down and fixed on a flow cell. This makes it possible to read out the respective break sequence in parallel. A four-step process was developed for this purpose.
1. Generation of the DNA - library
The DNA to be sequenced is broken down into random fragments using ultrasound. In order to sequence the fragments, they have to be fixed on a surface. For this purpose, each fragment is connected to an adapter on both sides. This adapter is a short special DNA double strand. These DNA fragments prepared in this way are amplified by a so-called PCR (polymerase chain reaction). PCR is a method that amplifies cellular DNA in the test tube.
2. DNA fixation and cluster generation
The amplified DNA double strands are split into single strands and transferred to a flow cell. This flow cell is coated with the two adapter strands as single strands. This leads to the fact that the prepared DNA single strand fragments can bind to the surface and thus form a double stranded adapter again. The fragments are distributed over the entire flow cell. In order to be able to later detect the incorporation of the bases specifically for each fragment, the fragments are cloned at their binding point using PCR (generation of identical copies). This creates points (clusters) that have bound a large amount to the same copy of DNA single strands. Each cluster can be analysed individually.
For the sequencing itself, the specific incorporation of a complementary base is detected by fluorescence signals. Each base resolves a specific light signal during integration. The entire flow cell is recorded and each cluster is a light spot. The light spots can be analysed according to their color sequence and thus reflect the sequence of the corresponding DNA fragment.
4. Data analysis
The reading of the color sequence of each light point and thus the sequence of the fragments is computer-based. After determining the fragment sequence, the sequences must be reassembled to the initial total DNA. For this purpose, overlaps of the fragments are used to reassemble them to the original DNA in their total length. This total DNA is compared with a reference DNA to detect changes in the sequence.
With this sequencing method, various questions can be answered.
Use of Next Generation Sequencing for PCDx Testing
As mentioned above, sequencing is a useful method for detecting changes in the genetic material of tumor cells. Paradigm analyses different changes on the DNA level depending on the type of cancer and known biomarkers.
The following changes in the genome are analysed in the PCDx test using Next Generation Sequencing:
Copy Number Variation (CNV)
DNA mutations, amplifications, insertions, deletions
Gene fusions within the entire genome
Microsatellite instability (MSI)
Tumor mutation burden (TMB)
Copy Number Variation (CNV)
Copy number variation is the variation in the number of genes present. In the classical sense, a gene is the genetic basis for a protein. Normally there are two genes for each protein, one inherited from the mother and the other from the father. However, in recent years it has been discovered that huge areas of the genome consist of repeats. These repetitions can be in areas where no gene is present, but genes themselves can also be present in quantities other than two or even missing. This possibility of variation in the number makes it possible for the organism to create another dimension of uniqueness, but a surplus or reduced presence can lead to disease. Copy Number Variation can be detected by Next Generation Sequencing, among other methods.
Today there is a lot of knowledge that links the present number of a specific gene with a disease, the effect of drugs or their response. For this reason, Paradigm uses the specific knowledge of relationships between CNV and tumor characteristics to propose an ideal therapy plan.
The DNA in a living organism is permanently exposed to DNA-damaging substances, such as UV rays in sunlight. Cells have developed several mechanisms to repair damaged DNA. However, this is not always successful and mutations occur. These are changes in the sequence of bases in the DNA. The base sequence is the code to make new proteins. If a base is permanently changed, it can lead to another protein with a different function or to the loss of its function. This is the main reason for the development of cancer. For example, a central cell death triggering protein can lose its effect and the cell suddenly becomes immortal, one of the main characteristics of the tumor cell.
However, only a very small number of mutations lead to pathological changes in the cells. Many mutations occur in DNA regions that do not encode a protein, or the mutation does not lead to a change in the structure and function of the resulting protein when the code is read. A huge field of research is concerned with the question of which mutations actually lead to the development of cancer. This knowledge helps to create specific therapies, as different cancer cells have certain weaknesses based on their underlying mutations.
Mutation is not only the change of a single base, but also amplifications, insertions and deletions belong to the category of mutation. In an amplification, a specific section of DNA is present in increased quantities. Insertion refers to the insertion of additional bases into the DNA. A deletion means that one or even more bases are missing.
The PCDx test analyses the tumor for various clinically validated mutations and then makes statements about possible treatment options. In order to be able to give meaningful recommendations for therapies, Paradigm works under the highest quality assurance requirements. Mutation analysis is only performed on tumor samples and Paradigm focuses on mutation analyses that result in drug treatment options or where clinical studies have proven that mutations are crucial for therapy.
The term gene fusion refers to the "fusing" of two genes that results from two previously separated genes. This can happen in a cell by chance during cell division. This results in the production of a new protein. As a result of the fusion, it is possible that the gene for protein A is linked to a piece of the gene for protein B, thereby producing protein A many times more. Other effects would be that a piece of protein B is built on protein A. This fusion can be positive for the cell but in many cases it is negative. The cell is a very complex mechanism that must be extremely finely tuned and by changing the amount of protein, the protein occurrence or the function, cancer cells can be created.
In 1960, for example, a cancer type was first associated with a specific fusion gene, the so-called Philadelphia chromosome in chronic myeloid leukaemia (CML). In this type of cancer, the proteins Bcl and Abl1 were fused together, resulting in a type of Abl1 protein with a permanent uncontrollable function. The altered Abl1 protein is constantly activated and can no longer be controlled by other proteins. As a result, the cell divides permanently and uncontrollably and cancer cells develop.
By recognizing fusion genes that lead to cancer cells, targeted therapies can be administered. In the case of Chronic Myeloid Leukemia (CML), which is based on the Philadelphia chromosome, special drugs (Imatinib) can be given which can inhibit the function of Abl1 and thus stop the uncontrolled division.
In the meantime, further fusion genes have been identified which cause cancer and for which there are suitable therapies. Therefore, the PCDx test also tests for these changes in order to better characterize the tumor and thus find the best therapy.
Tumor mutation burden (TMB)
When determining the tumor mutation burden, the number of mutations is determined for a defined amount of encoding genome (mutations per 1,000,000 DNA base pairs). Only those mutations that lead to a change at the protein level are counted here. Recent research has shown that the tumor mutation burden can be used as an indicator to predict whether a particular class of targeted anti-cancer drugs will have an effect against a particular tumor. This class of drugs are the immune checkpoint inhibitors. These drugs stimulate the immune system to recognize neoantigens and thus recognize and destroy the tumor cell as such. Neoantigen refers to the altered protein that results from the mutation and thus differs from the proteins in healthy cells.
Because of the shown correlation between tumor mutation burden and the level and effect of immune checkpoint inhibitors, the PCDx test detects the tumor mutation burden by means of next generation sequencing to create a further level of therapy prediction.
Microsatellite instability (MSI)
In DNA, short repeating (repetitive) sections occur, so-called microsatellites. In addition, certain proteins are present in a cell which repair DNA damage that has occurred. Such DNA damage can be caused by the duplication of DNA during cell division, which results in the incorporation of incorrect bases into the newly produced DNA strand ("mismatch"). If such repair proteins are themselves defective due to a mutation, this leads to new sections in the microsatellites in addition to further mutations in newly formed DNA. This leads to a change in length of the microsatellites. The detection of a microsatellite instability can then indirectly indicate a defect in the DNA repair system of the cell. Since there is a correlation between the presence of microsatellite instability and the response to immunotherapy, the PCDx test also tests for microsatellite instability.