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Mutations in Poly-G Repeats Enable Cancer Phylogeny Construction, Illuminating Disease Progression

COLD SPRING HARBOR, NY (GenomeWeb) — By tracing mutations in hypermutable polyguanine repeats located in intergenic regions, researchers have been able to reconstruct the phylogenies of patients' cancers, giving insight into how metastases develop.

Kamila Naxerova, a postdoctoral fellow at Harvard Medical School, said during a presentation at the Biology of Genomes conference here that she and her colleagues amplified these somatic poly-G stretches using PCR and determined their lengths by capillary electrophoresis to detect insertions and deletions that cropped up in those regions. Then by comparing mutational patterns of the poly-G repeats in primary tumors and at metastatic sites, the researchers built phylogenetic trees showing how the cancers progressed.

Just how metastases form and progress isn't yet clear, Naxerova noted. There are two main models for the process: early metastasis and parallel tumor evolution or late metastasis and cascading spread, she said, adding that intermediates between the two are also possible.

Knowing the process, she added, is important as the two models suggest different relationships between the primary and metastatic tumors. For instance, early metastasis and parallel tumor evolution predicts deep divergence between the primary and metastatic tumors while late metastasis and cascading spread of the cancer predicts that the lesions will be genetically similar to one another.

"Unfortunately, to date, there is not enough data to tell" which model is prevalent in various cancer types, Naxerova said.

Because these poly-G regions are hypermutable, Taq polymerase slippage occurs there during PCR amplification, generating a fragment distribution pattern that can be quantified down to single-base-pair resolution by capillary electrophoresis after PCR with fluorescent primers, as Naxerova and her colleagues recently published in the Proceedings of the National Academy of Sciences.

In a cohort of 22 colon cancer patients, Naxerova and her colleagues examined 20 somatic poly-G tracts, and were able to find poly-G variants in 91 percent of the patients' tumors. The patient samples, she said, had been formalin fixed and paraffin embedded.

Naxerova also noted that patient age is associated with the frequency of somatic mutations and, interestingly, that high-grade tumor samples had few mutations, a finding that they are delving into further.

Using these poly-G tract profiles, Naxerova and her colleagues constructed phylogenetic trees to determine how these tumors progressed in this set of patients.

For instance, one patient, a 66-year-old man, had a cecal tumor and a sigmoid tumor as well as metastases to the lymph node and liver. Naxerova genotyped samples from each location and constructed a tree. Based on the poly-G tract patterns, she found that the cecal and sigmoid tumors arose separately and then the sigmoid tumor gave rise to the lymph node and liver metastases.

Another patient, an 83-year-old woman, had tumors in her cecum and both ovaries, and Naxerova noted that pathological determination of whether the ovarian tumors arose separately or were metastases from the colon was difficult, though the pathologists decided they were indeed metastases.

The phylogenetic tree drawn using poly-G tract profiles from the cecum tumor samples and ovarian samples indicated that the colon tumor was much more diversified, though it did give rise to the more homogenous ovarian samples. This suggested to Naxerova that either a genetically advanced clone gave rise to both metastases or that one ovarian metastasis quickly gave rise to the other.

Then in a 48 year-old woman with adenocarcinoma of the colon, along with lymph node and ovarian metastases, the phylogenetic tree constructed from her poly-G tract profiles showed that the primary tumor and ovarian metastases were highly divergent. This, Naxerova said, could indicate an instance of parallel evolution.

However, the lymph node metastases were highly similar to the primary colon tumor, and Naxerova hypothesized that the primary tumor site could continually be seeding the lymph node metastases due to their close proximity.

Finally, analysis of tumor samples from a 44-year-old man with mucinous adenocarcinoma throughout his abdomen showed that all his tumors had similar poly-G patterns and a flat phylogenetic tree, indicating rapid clonal expansion.

Overall, Naxerova said that this approach of relying on mutations in poly-G repeats leads to robust phylogeny with minimal experimental effort and can be used on formalin-fixed, paraffin-embedded samples.