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 TNK2(P761Rfs*72) frameshift mutation was recurrent in EC, and the DDR1(R570Q) 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.

Pre-micro RNA signatures delineate stages of endothelial cell transformation in Kaposi sarcoma.

MicroRNAs (miRNA) have emerged as key regulators of cell lineage differentiation and cancer. We used precursor miRNA profiling by a novel real-time QPCR method (i) to define progressive stages of endothelial cell transformation cumulating in Kaposi sarcoma (KS) and (ii) to identify specific miRNAs that serve as biomarkers for tumor progression. We were able to compare primary patient biopsies to well-established culture and mouse tumor models. Loss of mir-221 and gain of mir-15 expression demarked the transition from merely immortalized to fully tumorigenic endothelial cells. Mir-140 and Kaposi sarcoma-associated herpesvirus viral miRNAs increased linearly with the degree of transformation. Mir-24 emerged as a biomarker specific for KS.

Tumor suppressor microRNAs are underrepresented in primary effusion lymphoma and Kaposi sarcoma.

The presence of tumor-specific microRNAs reflects tissue of origin and tumor stage. We show that the absence of miRNAs likewise can be used to determine tumor origin (miR-155) and proliferation state because tumor suppressor miRNAs (miR-222/221, let-7 family) were significantly down-regulated in primary effusion lymphoma (PEL) and in Kaposi sarcoma (KS), an endothelial cell tumor. PEL and KS are associated with KS-associated herpesvirus infection. We identified 15 virally regulated miRNAs in latently infected, nontumorigenic endothelial cells. MiR-143/145 were elevated only in KS tumors, not virally infected endothelial cells. Thus, they represent tumor-specific, rather than virus-specific, miRNAs. Because many tumor suppressor proteins are wild-type in KS and PEL, down-regulation of multiple tumor suppressor miRNAs provides a novel, alternative mechanism of transformation.

High-resolution copy number analysis of clear cell endometrial carcinoma.

Uterine cancer is the 6th leading cause of cancer death amongst American women. Most uterine cancers are endometrial carcinomas (ECs), which are classified into histological subtypes including endometrioid, serous, and clear cell ECs. Somatic copy number alterations (SCNAs) are frequent in serous EC, infrequent in endometrioid ECs, and poorly defined in clear cell ECs. The purpose of this study was to evaluate the occurrence of SCNAs in clinically diagnosed clear cell ECs. Paired tumor-normal DNAs for 51 ECs were hybridized to Illumina Infinium HumanHap650Y or Human660W-Quad Beadchips. Copy number calls were made using the Hidden Markov Model based SNP-FASST2 segmentation algorithm within Nexus Copy Number software (v.6.1). High-level SCNAs were defined as gain of ≥5 copies or homozygous deletion, both <10Mb. GISTIC 1.0, in Nexus, was used to identify statistically significant SCNAs, corrected for multiple testing. One or more high-level SCNAs were detected in 50% of 6 clear cell ECs, 78.6% of 28 serous ECs, and 17.6% of 17 endometrioid ECs. A positive association was found between high-level SCNAs and TP53 mutation across ECs (two-tailed p value<0.0001). Classifying tumors according to POLE, MSI, and TP53 status yielded four molecular subgroups; copy number altered tumors were more frequent in the TP53-mutated subgroup (95.8%) than in the unspecified subgroup (22.2%), and absent from the POLE and MSI subgroups. In conclusion, our study provides evidence of inter-tumor heterogeneity in the extent to which SCNAs occur in clinically diagnosed clear cell EC, and across molecular subgroups of EC. The co-occurrence of high-level SCNAs and TP53 mutations in some clear cell ECs is consistent with the view that a subset of clinically diagnosed clear cell ECs have molecular similarities to serous ECs.

The genomics and genetics of endometrial cancer.

Most sporadic endometrial cancers (ECs) can be histologically classified as endometrioid, serous, or clear cell. Each histotype has a distinct natural history, clinical behavior, and genetic etiology. Endometrioid ECs have an overall favorable prognosis. They are typified by high frequency genomic alterations affecting PIK3CA, PIK3R1, PTEN, KRAS, FGFR2, ARID1A (BAF250a), and CTNNB1 (ß-catenin), as well as epigenetic silencing of MLH1 resulting in microsatellite instability. Serous and clear cell ECs are clinically aggressive tumors that are rare at presentation but account for a disproportionate fraction of all endometrial cancer deaths. Serous ECs tend to be aneuploid and are typified by frequent genomic alterations affecting TP53 (p53), PPP2R1A, HER-2/ERBB2, PIK3CA, and PTEN; additionally, they display dysregulation of E-cadherin, p16, cyclin E, and BAF250a. The genetic etiology of clear cell ECs resembles that of serous ECs, but it remains relatively poorly defined. A detailed discussion of the characteristic patterns of genomic alterations that distinguish the three major histotypes of endometrial cancer is reviewed herein.

Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes.

Endometrial cancer is the sixth most commonly diagnosed cancer in women worldwide, causing ~74,000 deaths annually. Serous endometrial cancers are a clinically aggressive subtype with a poorly defined genetic etiology. We used whole-exome sequencing to comprehensively search for somatic mutations within ~22,000 protein-encoding genes in 13 primary serous endometrial tumors. We subsequently resequenced 18 genes, which were mutated in more than 1 tumor and/or were components of an enriched functional grouping, from 40 additional serous tumors. We identified high frequencies of somatic mutations in CHD4 (17%), EP300 (8%), ARID1A (6%), TSPYL2 (6%), FBXW7 (29%), SPOP (8%), MAP3K4 (6%) and ABCC9 (6%). Overall, 36.5% of serous tumors had a mutated chromatin-remodeling gene, and 35% had a mutated ubiquitin ligase complex gene, implicating frequent mutational disruption of these processes in the molecular pathogenesis of one of the deadliest forms of endometrial cancer.

Gene alteration and precursor and mature microRNA transcription changes contribute to the miRNA signature of primary effusion lymphoma.

MicroRNAs are regulated by gene alteration, transcription, and processing. Thus far, few studies have simultaneously assessed all 3 levels of regulation. Using real-time quantitative polymerase chain reaction (QPCR)-based arrays, we determined changes in gene copy number, pre-miRNA, and mature miRNA levels for the largest set of primary effusion lymphomas (PELs) to date. We detected PEL-specific miRNA gene amplifications, and concordant changes in pre-miRNA and mature miRNA. We identified 68 PEL-specific miRNAs. This defines the miRNA signature of PEL and shows that transcriptional regulation of pre-miRNA as well as mature miRNA levels contribute nonredundant information that can be used for the classification of human tumors.