Second generation sequencing of the mesothelioma tumor genome

Our plain-English summary of Second generation sequencing of the mesothelioma tumor genome, by R. Bueno, A. De Rienzo, L. Dong, et al (PloS One, 2010).

The study

• Authors: R. Bueno, A. De Rienzo, L. Dong, G.J. Gordon, C.F. Hercus, W.G. Richards, R.V. Jensen, A. Anwar, G. Maulik, L.R. Chirieac, K.F. Ho, B.E. Taillon, C.L. Turcotte, R.G. Hercus, S.R. Gullans, D.J. Sugarbaker
• Journal: PloS One
• Published: 2010;5(5):e10612
• Cited by: 54 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The current paradigm for elucidating the molecular etiology of cancers relies on the interrogation of small numbers of genes, which limits the scope of investigation. Emerging second-generation massively parallel DNA sequencing technologies have enabled more precise definition of the cancer genome on a global scale. We examined the genome of a human primary malignant pleural mesothelioma (MPM) tumor and matched normal tissue by using a combination of sequencing-by-synthesis and pyrosequencing methodologies to a 9.6X depth of coverage. Read density analysis uncovered significant aneuploidy and numerous rearrangements. Method-dependent informatics rules, which combined the results of different sequencing platforms, were developed to identify and validate candidate mutations of multiple types. Many more tumor-specific rearrangements than point mutations were uncovered at this depth of sequencing, resulting in novel, large-scale, inter- and intra-chromosomal deletions, inversions, and translocations. Nearly all candidate point mutations appeared to be previously unknown SNPs. Thirty tumor-specific fusions/translocations were independently validated with PCR and Sanger sequencing. Of these, 15 represented disrupted gene-encoding regions, including kinases, transcription factors, and growth factors. One large deletion in DPP10 resulted in altered transcription and expression of DPP10 transcripts in a set of 53 additional MPM tumors correlated with survival. Additionally, three point mutations were observed in the coding regions of NKX6-2, a transcription regulator, and NFRKB, a DNA-binding protein involved in modulating NFKB1. Several regions containing genes such as PCBD2 and DHFR, which are involved in growth factor signaling and nucleotide synthesis, respectively, were selectively amplified in the tumor. Second-generation sequencing uncovered all types of mutations in this MPM tumor, with DNA rearrangements representing the dominant type.

In plain English

This laboratory study, which the authors called pilot work, used whole-genome sequencing to examine one malignant pleural mesothelioma tumor. The tumor was removed during surgery from a male patient at Brigham and Women's Hospital in Boston. The authors compared it with normal lung tissue from the same man, using two different sequencing technologies. The article does not report this patient's asbestos exposure. The tumor showed large-scale gains and losses of chromosome material. The authors confirmed 30 tumor-specific DNA rearrangements, 15 of which disrupted 17 genes, and three point mutations, in the genes NKX6-2, CDH8 and NFRKB. None of the 30 rearrangements was found at the same breakpoints in 53 other mesothelioma tumors. The DPP10 gene was partly deleted in the first tumor. Among the 53 other patients, those whose tumors had any detectable DPP10 transcript (the RNA copy of the gene) had a median survival of 22 months, against 8 months for the others (P = 0.004). The authors concluded that this single tumor contained many major tumor-specific rearrangements. They noted that the genome sequencing focused on a tumor from a single patient, and that each sequencing method has a large number of false-positive artifacts.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Whole-genome sequencing of a single tumor and matched normal tissue. This was a laboratory genetic study, described by the authors as pilot work, with follow-up tests in 53 additional tumors and in previously published gene-activity data from 40 tumors.
• Who was studied: One male patient with a mixed-histology malignant pleural mesothelioma undergoing surgery at Brigham and Women's Hospital, Boston, with normal lung tissue from the same person. The study also used 53 additional malignant pleural mesothelioma tumors from the institution's tumor bank, and published microarray data from 40 malignant pleural mesothelioma patients.
• Exposure: Not studied. The article does not report asbestos exposure or any other exposure for the patient or the additional tumors. It examined genetic changes in the tumor.
• Main results:
  • Combining the two technologies, the tumor genome was sequenced to nearly 10X total coverage.
  • The tumor showed large-scale aneuploidy (abnormal numbers of chromosome copies). Sequencing results matched an independent method, comparative genomic hybridization arrays, with a Pearson correlation coefficient of 0.7918.
  • In the patient's tumor, 30 tumor-specific rearrangements were validated: 6 between chromosomes and 24 within chromosomes. Of these, 15 disrupted 17 genes.
  • None of the 30 rearrangements was found at the exact same breakpoints in the 53 additional mesothelioma tumors tested.
  • One rearrangement deleted a large part of the DPP10 gene in the patient's tumor. In the 53 additional tumors, DPP10 transcript was detected in 31 (55%), and truncated transcripts were found in 7 of those 31.
  • Among the 53 additional patients, those whose tumors expressed any DPP10 transcript had statistically significantly better overall survival than those whose tumors did not: median survival 22 months versus 8 months.
  • Of 89 candidate point mutations checked by Sanger sequencing, 83 were heterozygous novel SNPs (inherited variants) and 3 were homozygous in the tumor only. The other 3 were heterozygous point mutations in NKX6-2, CDH8 and NFRKB. The NKX6-2 and NFRKB mutations changed protein-coding sequence; the CDH8 mutation was within an intron.
  • In the tumor, 14 regions with rearrangements were amplified more than 5-fold compared with normal DNA. These regions included the DHFR and PCBD2 genes.
• The authors' conclusion: The authors concluded that this single mesothelioma tumor had many major tumor-specific chromosomal rearrangements. They also concluded that clues from one tumor's sequence can lead to genes that might be inactivated in other ways in related tumors. They considered two steps essential, given the many polymorphisms and sequencing artifacts: comparing tumor with normal sequence, and confirming with a second sequencing method.
• Limitations the authors state:
  • The work focused on a tumor from a single patient, and the sequencing depth may have been biased toward finding rearrangement-type mutations.
  • Each sequencing technology used is reported to produce many false-positive artifacts, so neither may be accurate enough on its own.
  • Functional analysis, deeper sequencing and genotyping in more specimens are needed to identify true driver mutations in mesothelioma.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelioma, Etiology, Pleural Mesothelioma, Translocation.

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