Mutational analysis of the tyrosine kinome in serous and clear cell endometrial cancer uncovers rare somatic mutations in TNK2 and DDR1

Background: Endometrial cancer (EC) is the 8th leading cause of cancer death amongst American women. Most ECs are endometrioid, serous, or clear cell carcinomas, or an admixture of histologies. Serous and clear ECs are clinically aggressive tumors for which alternative therapeutic approaches are needed. The purpose of this study was to search for somatic mutations in the tyrosine kinome of serous and clear cell ECs, because mutated kinases can point to potential therapeutic targets. Methods: In a mutation discovery screen, we PCR amplified and Sanger sequenced the exons encoding the catalytic domains of 86 tyrosine kinases from 24 serous, 11 clear cell, and 5 mixed histology ECs. For somatically mutated genes, we next sequenced the remaining coding exons from the 40 discovery screen tumors and sequenced all coding exons from another 72 ECs (10 clear cell, 21 serous, 41 endometrioid). We assessed the copy number of mutated kinases in this cohort of 112 tumors using quantitative real time PCR, and we used immunoblotting to measure expression of these kinases in endometrial cancer cell lines. Results: Overall, we identified somatic mutations in TNK2 (tyrosine kinase non-receptor, 2) and DDR1 (discoidin domain receptor tyrosine kinase 1) in 5.3% (6 of 112) and 2.7% (3 of 112) of ECs. Copy number gains of TNK2 and DDR1 were identified in another 4.5% and 0.9% of 112 cases respectively. Immunoblotting confirmed TNK2 and DDR1 expression in endometrial cancer cell lines. Three of five missense mutations in TNK2 and one of two missense mutations in DDR1 are predicted to impact protein function by two or more in silico algorithms. The TNK2P761Rfs*72 frameshift mutation was recurrent in EC, and the DDR1R570Q missense mutation was recurrent across tumor types. Conclusions: This is the first study to systematically search for mutations in the tyrosine kinome in clear cell endometrial tumors. Our findings indicate that high-frequency somatic mutations in the catalytic domains of the tyrosine kinome are rare in clear cell ECs. We uncovered ten new mutations in TNK2 and DDR1 within serous and endometrioid ECs, thus providing novel insights into the mutation spectrum of each gene in EC

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Abstract 5312: Mutational analysis of the tyrosine kinome in serous and clear cell endometrial cancers.

Abstract Endometrial cancers (ECs) arise from the inner epithelial lining of the uterus. At diagnosis, the majority of ECs are carcinomas of endometrioid, serous, or clear cell histologies, or an admixture of at least two of these histological subtypes. Serous and clear ECs are rare at presentation but contribute disproportionately to mortality from EC. Thus, there is an acute need for novel therapeutic approaches to treat serous and clear cell ECs. Small molecule inhibitors directed against tyrosine kinases have shown clinical efficacy in other types of cancer in which the target kinase is mutationally activated. Here, we used Sanger sequencing to systematically search for somatic mutations within 527 exons that encode the catalytic domains of 85 tyrosine kinases from 23 serous, 10 clear cell, and 5 mixed histology ECs. For mutated genes, we extended the screen to another 73 ECs (21 serous, 10 clear cell, 42 endometrioid). Genes that were mutated in both the primary and secondary screens were resequenced in a tertiary screen to search for mutations in all coding exons among the entire panel of 111 endometrial tumors. Overall, we uncovered nonsynonymous somatic mutations of ACK1, DDR1, and KDR in 5.4%, 3.6%, and 1% of ECs. Serous tumors had mutated ACK1, DDR1 and KDR in 2.3%, 4.5%, and 2.3% of cases. Clear cell tumors had mutated ACK1 in 5% of cases. Endometrioid tumors had mutated DDR1 in 2.3% of cases. Immunoblotting confirmed that ACK1 and DDR1 are endogenously expressed in endometrial cancer cell-lines. Eighty-seven percent (7 of 8) of ACK1 mutations and 50% (2 of 4) of DDR1 mutations are predicted to impact protein function. To our knowledge this is the first report of ACK1 and DDR1 mutations in endometrial cancer. Our findings warrant future studies to determine whether the ACK1 and DDR1 mutations uncovered here are pathogenic. Citation Format: Meghan L. Rudd, Hassan Mohamed, Jessica C. Price, Mary Ellen Urick, Andrea J. O'Hara, Matthieu Le Gallo, NISC Comparative Sequencing Program, Pedro Cruz, Suiyuan Zhang, James Mullikin, Maria J. Merino, Daphne W. Bell. Mutational analysis of the tyrosine kinome in serous and clear cell endometrial cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5312. doi:10.1158/1538-7445.AM2013-5312</jats:p

Sequencing of Candidate Chromosome Instability Genes in Endometrial Cancers Reveals Somatic Mutations in <i>ESCO1</i>, <i>CHTF18</i>, and <i>MRE11A</i>

<div><p>Most endometrial cancers can be classified histologically as endometrioid, serous, or clear cell. Non-endometrioid endometrial cancers (NEECs; serous and clear cell) are the most clinically aggressive of the three major histotypes and are characterized by aneuploidy, a feature of chromosome instability. The genetic alterations that underlie chromosome instability in endometrial cancer are poorly understood. In the present study, we used Sanger sequencing to search for nucleotide variants in the coding exons and splice junctions of 21 candidate chromosome instability genes, including 19 genes implicated in sister chromatid cohesion, from 24 primary, microsatellite-stable NEECs. Somatic mutations were verified by sequencing matched normal DNAs. We subsequently resequenced mutated genes from 41 additional NEECs as well as 42 endometrioid ECs (EECs). We uncovered nonsynonymous somatic mutations in <i>ESCO1</i>, <i>CHTF18,</i> and <i>MRE11A</i> in, respectively, 3.7% (4 of 107), 1.9% (2 of 107), and 1.9% (2 of 107) of endometrial tumors. Overall, 7.7% (5 of 65) of NEECs and 2.4% (1 of 42) of EECs had somatically mutated one or more of the three genes. A subset of mutations are predicted to impact protein function. The co-occurrence of somatic mutations in <i>ESCO1</i> and <i>CHTF18</i> was statistically significant (<i>P</i> = 0.0011, two-tailed Fisher's exact test). This is the first report of somatic mutations within <i>ESCO1</i> and <i>CHTF18</i> in endometrial tumors and of <i>MRE11A</i> mutations in microsatellite-stable endometrial tumors. Our findings warrant future studies to determine whether these mutations are driver events that contribute to the pathogenesis of endometrial cancer.</p></div

Localization of somatic mutations in <i>ESCO1</i>, <i>CHTF18</i>, and <i>MRE11A</i> in primary endometrial tumors, relative to important functional domains of the encoded proteins.

<p>Individual somatic mutations are indicated by squares (nonsense mutations) or diamonds (missense mutations). Domain positions are derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063313#pone.0063313-Berkowitz1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063313#pone.0063313-Park1" target="_blank">[66]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063313#pone.0063313-Moreau1" target="_blank">[61]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063313#pone.0063313-Stracker1" target="_blank">[59]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063313#pone.0063313-Williams1" target="_blank">[67]</a>. GAR: Glycine-Arginine-Rich motif; RBD:RAD50 Binding Domain; RFC box: Replication Factor C box.</p

Oncoprint displaying nonsynonymous somatic mutations in <i>ESCO1</i>, <i>CHTF18</i>, <i>MRE11A</i>, and <i>ATAD5</i> in eight primary endometrial cancers.

<p>Individual tumors (T) are indicated by vertical gray bars. Tumors consist of NEECs (T3, T51, T62, T68, T77, T79, T113) and an EEC (T88). Genes (left) and nonsynonymous somatic mutations (orange boxes) are indicated. <i>ESCO1</i>, <i>CHTF18</i>, and <i>MRE11A</i> were analyzed in this study; *<i>ATAD5</i> mutations, <i>MSH6</i> mutations, and microsatellite instability (MSI) have previously been described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063313#pone.0063313-Bell1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063313#pone.0063313-LeGallo1" target="_blank">[52]</a>.</p