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Two Sequencing Studies ID Frequently Mutated Gene in Ovarian Cancer Subtype

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By Monica Heger

This article has been updated from a version posted Sept. 9 to include outside comment..

In the search for cancer-causing mutations, sequencing studies have mostly identified genes mutated in a small proportion of cases, with a smoking gun remaining elusive.

But now, two separate research groups have used sequencing to identify a novel, commonly mutated gene in ovarian clear-cell carcinoma. The gene was mutated in about half of all cases, making it a promising candidate for diagnostics and therapeutics.

Researchers from Johns Hopkins University published the results of a whole-exome study in Science last week, while a separate group from the BC Cancer Agency used transcriptome sequencing to come to the same conclusions. Their study was published last week in the New England Journal of Medicine.

"There are very few genes in cancer that have been found to be mutated in high proportion," Nickolas Papadopoulos, director of translational genetics at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University and a senior author of the Science paper, told In Sequence. "When a finding like this happens, it's exciting because of its novelty."

"Whenever there's a high prevalent, recurrent mutation it suggests that it is biologically important for tumor progression," said Arul Chinnaiyan, director of the Michigan Center for Translational Pathology, who was not involved with either study, but who has used transcriptome sequencing to search for druggable mutations in cancer (IS 6/8/2010).

Chinnaiyan added that it was "reassuring" that the two different methods came to the same conclusion. "It might be expected, but it's nice to see that they came to the same results using independent methods."

He also said that while both methods would be less comprehensive than whole-genome sequencing, they both avoid the problem of generating too much data. "Sometimes you get lost in all the additional information and might miss the low-hanging fruit," he said.

Johns Hopkins' Papadopoulos and his team sequenced the exomes of eight tumor samples from patients with ovarian clear cell carcinoma to an average 84-fold coverage, using Agilent's SureSelect target enrichment and the Illumina Genome Analyzer.

They identified 268 somatic mutations in 253 genes and confirmed 237 of them by Sanger sequencing. Four genes — PIK3CA, KRAS, PPP2R1A, and ARID1A — were mutated in more than one of the eight tumors.

The team then sequenced those four genes from matched tumor/normal DNA in 34 additional cases using Sanger sequencing.

In total, mutations in PIK3CA, KRAS, PPP2R1A, and ARID1A were identified in 40 percent, 4.7 percent, 7.1 percent, and 57 percent of the 42 tumors, respectively. Both PIK3CA and KRAS have been previously implicated in ovarian cancer and are well-characterized, but PP2R1A and ARID1A are novel. The researchers hypothesize that PPP2R1A is an oncogene, while ARID1A is a tumor suppressor. Oncogenes tend to have mainly missense mutations all at the same codon, or clustered at codons adjacent to each other, while tumor suppressors tend to be mutated at a variety of different positions in the coding region, and the mutations typically truncate the encoded protein. Also, tumor suppressors tend to affect both alleles, while oncogenes often only affect one. The mutations in PPP2R1A were clustered, while mutations occurred throughout ARID1A and were predicted to truncate the protein.

Papadopoulos said the team would now focus on understanding the function of ARID1A. "We know it's involved in a complex of proteins that remodels the chromatin, which packages DNA. And this packaging of the DNA has implications in which a whole series of genes are regulated," he said.

He said his team decided to use whole-exome sequencing because focusing on the protein-coding region would provide "more of an immediate chance to develop clinical applications." He added that for future studies, they would continue to do exome sequencing. "Whole-genome sequencing has its advantages, but we're not going to abandon exome sequencing," he said.

Meanwhile, the group from BC Cancer Agency independently obtained similar results using transcriptome sequencing. They sequenced the transcriptomes of 18 ovarian clear-cell carcinoma tumors and one cell line, using paired-end sequencing on the Illumina GA. When they began the study, they were sequencing with read lengths of 37 base pairs, but by the end, they had increased to 75 base pairs.

David Huntsman, a genetic pathologist at the BC Cancer Agency and senior author of the paper, said that transcriptome sequencing provided a "very rich data set. The appeal is that you get mutations not only in coding genes but also gene fusions, and also accurate gene expression." However, he added, while it is a rich data set, it is imperfect and does not always catch every mutation. "If you marry transcriptome sequencing to exome or whole-genome sequencing, then you have a data set which is very well rounded."

Huntsman and his team found mutations in the ARID1A gene in six samples. They then used a targeted exon resequencing strategy to sequence the gene in an additional 210 samples, including 101 samples from patients with clear-cell carcinoma, 33 samples from patients with endometrioid carcinoma, and 76 samples from patients with high-grade serous carcinoma.

They found mutations in 46 percent of the ovarian clear-cell carcinoma patients, 30 percent of the endometrioid carcinomas, and none of the high-grade serous ovarian carcinomas.

The team also did an immunohistochemical analysis in more than 400 additional ovarian cancer tumors for the protein BAF250a, which is encoded by ARID1A and a key component of the chromatin remodeling complex. Loss of expression was strongly correlated with ARID1A mutations in both ovarian clear-cell carcinoma and endometrioid carcinoma, but not high-grade serous carcinoma.

The high-grade serous carcinoma subtype is being sequenced by the Cancer Genome Atlas project, but the current study's finding that ARID1A does not play a role in that subtype appears to explain why the gene had not been previously implicated in ovarian cancer. Major disruption of genomic integrity is a key feature in that subtype, Huntsman said, but not in the clear-cell carcinoma subtype. Huntsman speculated that ARID1A mutations might define ovarian clear-cell carcinoma, and other cancer subtypes not marked by major genomic instability.

Huntsman added that the identification of ARID1A mutations in endometrioid cancer suggests that the gene could be used as a biomarker to determine which women with endometriosis are at the greatest risk for developing cancer. "Only a tiny fraction of women with endometriosis ever develop cancer," he said. "Having a better tool to identify which women are at risk could help guide therapy" and determine which women would most benefit from surgery.

Both Huntsman and Papapdopoulos said that it would be difficult to target the ARID1A gene directly with a drug because it is a tumor suppressor, so mutations in the gene cause it to lose function.

"It's easier to have a protein that is still active and [use drugs to] try to prevent its activity, rather than to have something missing and try to substitute for it," Papadopoulos said. As a result, a major next step will be to determine which genes are regulated by ARID1A, he added, and then to figure out which of those are druggable, or whether there are already drugs that target any of them.

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