Recent advances in DNA sequencing technology allow, for the first time, identification of tumor-specific gene mutations for each tumor at a genome-wide level. Most such mutations are acquired accidentally during lifetime, for example, during cell division or from exposure to environmental factors. If a cell acquires too many mutations in genes that are critical for growth and/or differentiation, the cell will be transformed into a malignant or cancer cell. If this happens to a blood-forming cell in bone marrow the result is leukaemia. There is increasing evidence that, although cancers may look identical to a pathologist, they are genetically highly diverse and each cancer is, therefore, a “malignant individual” (“heimtückisches Einzelwesen“).
A research team led by me and Stefan Bohlander from the Department of Internal Medicine 3 at the Ludwig-Maximilians-Universität (LMU) in Munich is working towards the identification of all genetic alterations of some specific subtypes of leukaemia. Such a complete listing of all mutations in a specific type of cancer is known as a “mutatome”.
The Result of Several Genetic Changes
As only about two percent of the human genome is used to specify the amino acid sequence of our proteins and as only one third of the 23.000 human genes are active in blood cells, the research team chose to focus on active genes. To look for mutations only in active genes is a faster and more economical approach than to search through the entire genome which contains a lot of non-coding DNA sequences whose function is largely unknown.
“Although, the complexity of different mutations and their possible combinations is overwhelming, we have now the tools in hand to systematically analyze all active genes in every cancer sample.” says Bohlander. Using next-generation sequencing technology, the team has detected numerous genetic changes in a sample from a single leukaemia patient. The changes included a mutation in the RUNX1 gene, which is reported to be frequently altered in a certain type of blood cancer. Interestingly, they also found a mutation in the TLE4 gene, which normally cooperates with the RUNX1 gene; both genes encode proteins that bind to each other. These findings suggest that a malignancy is the result of several genetic changes, with each change bringing the cell a small step closer to malignant status, rather than the consequence of a single mutation producing a large effect on the cell.
Comparing Blood Samples
The first complete analysis of a human genome took 13 years (1990–2003) and an estimated expenditure of about US$ 2 billion. In 2012, sequencing a human genome costs about 20.000 US$, while sequencing only the protein-coding regions or only the active genes costs about 1.000 US$. In 2008, the first human-leukaemia genome was analyzed. Despite the use of the latest sequencing technologies, the project took several years and an expenditure of several million US dollars. Although many mutations were found in that genome, their relevance remained unclear because all of the newly observed mutations were only found in the original sample. None of the corresponding genes in the more than 180 additional leukaemia samples that were analyzed showed any such mutations.
To identify tumor-specific mutations it is essential to compare the tumor sample to a healthy tissue sample from the same patient. Rather than examining only a specific list of genes which had previously been shown to be mutated in a specific tumor type, it is possible, following successful chemotherapy, to compare blood samples that are devoid of leukaemia cells with pre-chemotherapy leukaemia cells. Using this approach it is possible to identify all tumor-specific mutations in the active genes. This approach constitutes a quantum leap in tumor analysis, since examining only those genes that had previously been linked to a specific tumor type prevents the detection of additional genes that are relevant to specific tumors.
Genetic Diversity as a Massive Challenge
A key challenge to the provision of personalized medicine is to determine individual genetic alterations in each tumor. Such genetic alterations can then be used as predictive or prognostic biomarkers and also as molecular targets for new therapies. Our research team is confident that patients will soon benefit from the diagnostic application of this innovative type of mutation screening, applications that consider the presence or absence of certain mutations to predict the course of the disease and its response to therapies.
However, the development of truly personalized therapies is still a massive challenge because of the enormous genetic diversity in the malignant individuals. Although many current, state-of-the-art chemotherapies are efficient in the treatment of cancer they are not specific to the malignant individuals and also kill non-malignant cells. Even though identifying new tumor-specific mutations will not immediately result in new therapies, it will at least provide for a better understanding of our enemy, the malignant individuals.
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This research project is funded by a German Cancer Aid grant (109031) to Philipp A. Greif and Stefan K. Bohlander, and a grant from the BMBF to Stefan K. Bohlander (NGFN Plus, PKL-01-GS0876-6).