First Sequencing of Cancer Patient's Genome Completed

A large research team has sequenced the genome of the patient--a woman in her 50s who ultimately died of her disease--and the genome of her leukemia cells, to identify genetic changes unique to her cancer.

The study, reported in the November 6, 2008, issue of the journal Nature, was conducted by scientists from the Genome Sequencing Center and the Siteman Cancer Center at Washington University School of Medicine in St. Louis (MO, USA; http://medicine.wustl.edu).

The groundbreaking project sets the stage for using a more comprehensive, genome-wide approach to unravel the genetic basis of cancer. "Our work demonstrates the power of sequencing entire genomes to discover novel cancer-related mutations,” commented senior author Richard K. Wilson, Ph.D., director of Washington University's Genome Sequencing Center. "A genome-wide understanding of cancer, which is now possible with faster, less expensive DNA sequencing technology, is the foundation for developing more effective ways to diagnose and treat cancer.”

The researchers discovered just 10 genetic mutations in the patient's tumor DNA that appeared to be relevant to her disease; eight of the mutations were rare and occurred in genes that had never been linked to AML. They also showed that virtually every cell in the tumor sample had nine of the mutations, and that the single genetic alteration that occurred less frequently was likely the last to be acquired. The scientists suspect that all the mutations were important to the patient's cancer.

Similar to most cancers, AML--a cancer of blood-forming cells in the bone marrow--arises from mutations that accumulate in people's DNA over the course of their lives. However, little is known about the precise nature of those changes and how they disrupt biologic pathways to cause the uncontrolled cell growth that is the key characteristic of cancer.

Previous efforts to decode individual human genomes have looked at common points of DNA variation that may be relevant for disease risk. What is remarkable about the new research is that the scientists were able to sift through the 3 billion pairs of chemical bases that make up the human genome to pull out the mutations that contributed to the patient's cancer. "Until now, no one has sequenced a patient's genome to find all the mutations that are unique to that person's disease,” said lead author Timothy Ley, M.D., a hematologist and professor of medicine. "We didn't know what we would find, but we felt that the answers to why this patient had AML had to be embedded in her DNA.”

Up to now, scientists involved in large-scale genetic studies of cancer have not gone so far as to do a full side-by-side comparison of the genomes of normal cells and tumor cells from the same patient. Instead, the majority of earlier studies have involved the sequencing of genes with known or suspected relationships to cancer, a method that likely misses key mutations.

Based on genetic testing with traditional methods at the study's outset, the patient was known to have two mutations that are common among AML patients, an indicator she had a typical subtype of the disease, and one of the many reasons why her genome was selected for sequencing. The researchers sequenced the patient's full genome, meaning DNA from both sets of chromosomes, using genetic material obtained from a skin sample. This gave the scientists a reference DNA sequence to which they could compare genetic alterations in the patient's tumor cells, taken from a bone marrow sample that was comprised only of tumor cells. Both samples were obtained before the patient received cancer treatment, which can further damage DNA.

The scientists then looked for genetic differences--points of single base changes in the DNA--in the patient's tumor genome compared with her normal genome. Of the nearly 2.7 million single nucleotide variants in the patient's tumor genome, almost 98% also were detected in DNA from the patient's skin sample, thereby narrowing the number of variants that required further study to about 60,000.

Using advanced software and analytic tools, some of which the researchers developed specifically for this project, the scientists identified the 10 mutations (including the two previously known genetic mutations that are common to her leukemia subtype but do not directly cause the disease) by looking for single base DNA changes that altered the instructions for making proteins.

Of the eight novel mutations discovered, three were found in genes that normally act to suppress tumor growth. One of these mutations is in the PTPRT tyrosine phosphatase gene, which is frequently altered in colon cancer. Four other mutated genes appear to be involved in molecular pathways that promote cancer growth. Specifically, one mutation was found in a gene family that also is expressed in embryonic stem cells and may be involved with cell self-renewal. Interestingly, the researchers note, self-renewal is believed to be a fundamental characteristic of leukemia cells.

Another gene alteration appears to affect the transport of drugs into the cell, and may have contributed to the patient's chemotherapy resistance. "We're still analyzing the patient's non-coding DNA and expect to find a number of additional relevant mutations in this portion of the genome,” stated Elaine Mardis, Ph.D., co-lead author of the study and codirector of the Genome Sequencing Center. "But the role of these noncoding mutations will be more of a challenge to elucidate because we do not yet fully understand the function of this part of the genome.”

The team also looked to see if the eight novel mutations in the patient's tumor genome also occurred in the DNA of tumor samples from 187 additional AML patients. None of those tumors had any of the eight mutations. "This suggests that there is a tremendous amount of genetic diversity in cancer, even in this one disease,” Dr. Wilson said. "There are probably many, many ways to mutate a small number of genes to get the same result, and we're only looking at the tip of the iceberg in terms of identifying the combinations of genetic mutations that can lead to AML.”

Based on their current understanding of cancer, the researchers suspect that the mutations occurred sequentially. The first mutation gave the cell a slight tendency toward cancer, and then one by one, the other genetic alterations were acquired, with each contributing something to the cancer. One mutation, in the FLT3 gene, was not present in all of the tumor cells, and they suspect that it was the last one to occur. "The final mutation may represent a tipping point that causes the cancer cells to become more dangerous,” Dr. Ley remarked.

The team is now sequencing the genomes of additional patients with AML, and they are planning to expand the whole-genome approach to breast and lung cancers.

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