Kyle Covington

Trans-ancestry mutational landscape of hepatocellular carcinoma genomes

Diverse epidemiological factors are associated with hepatocellular carcinoma (HCC) prevalence in different populations. However, the global landscape of the genetic changes in HCC genomes underpinning different epidemiological and ancestral backgrounds still remains uncharted. Here a collection of data from 503 liver cancer genomes from different populations uncovered 30 candidate driver genes and 11 core pathway modules. Furthermore, a collaboration of two large-scale cancer genome projects comparatively analyzed the trans-ancestry substitution signatures in 608 liver cancer cases and identified unique mutational signatures that predominantly contribute to Asian cases. This work elucidates previously unexplored ancestry-associated mutational processes in HCC development. A combination of hotspot TERT promoter mutation, TERT focal amplification and viral genome integration occurs in more than 68% of cases, implicating TERT as a central and ancestry-independent node of hepatocarcinogenesis. Newly identified alterations in genes encoding metabolic enzymes, chromatin remodelers and a high proportion of mTOR pathway activations offer potential therapeutic and diagnostic opportunities.

Comprehensive Genomic Analysis Identifies Novel Subtypes and Targets of Triple-Negative Breast Cancer

Purpose: Genomic profiling studies suggest that triple-negative breast cancer (TNBC) is a heterogeneous disease. In this study, we sought to define TNBC subtypes and identify subtype-specific markers and targets. Experimental Design: RNA and DNA profiling analyses were conducted on 198 TNBC tumors [estrogen receptor (ER) negativity defined as Allred scale value ≤ 2] with >50% cellularity (discovery set: n = 84; validation set: n = 114) collected at Baylor College of Medicine (Houston, TX). An external dataset of seven publically accessible TNBC studies was used to confirm results. DNA copy number, disease-free survival (DFS), and disease-specific survival (DSS) were analyzed independently using these datasets. Results: We identified and confirmed four distinct TNBC subtypes: (i) luminal androgen receptor (AR; LAR), (ii) mesenchymal (MES), (iii) basal-like immunosuppressed (BLIS), and (iv) basal-like immune-activated (BLIA). Of these, prognosis is worst for BLIS tumors and best for BLIA tumors for both DFS (log-rank test: P = 0.042 and 0.041, respectively) and DSS (log-rank test: P = 0.039 and 0.029, respectively). DNA copy number analysis produced two major groups (LAR and MES/BLIS/BLIA) and suggested that gene amplification drives gene expression in some cases [FGFR2 (BLIS)]. Putative subtype-specific targets were identified: (i) LAR: androgen receptor and the cell surface mucin MUC1, (ii) MES: growth factor receptors [platelet-derived growth factor (PDGF) receptor A; c-Kit], (iii) BLIS: an immunosuppressing molecule (VTCN1), and (iv) BLIA: Stat signal transduction molecules and cytokines. Conclusion: There are four stable TNBC subtypes characterized by the expression of distinct molecular profiles that have distinct prognoses. These studies identify novel subtype-specific targets that can be targeted in the future for the effective treatment of TNBCs. Clin Cancer Res; 21(7); 1688–98. ©2014 AACR. See related commentary by Vidula and Rugo, p. 1511

Mutually exclusive recurrent somatic mutations in MAP2K1 and BRAF support a central role for ERK activation in LCH pathogenesis.

Langerhans cell histiocytosis (LCH) is a myeloproliferative disorder characterized by lesions composed of pathological CD207(+) dendritic cells with an inflammatory infiltrate. BRAFV600E remains the only recurrent mutation reported in LCH. In order to evaluate the spectrum of somatic mutations in LCH, whole exome sequencing was performed on matched LCH and normal tissue samples obtained from 41 patients. Lesions from other histiocytic disorders, juvenile xanthogranuloma, Erdheim-Chester disease, and Rosai-Dorfman disease were also evaluated. All of the lesions from histiocytic disorders were characterized by an extremely low overall rate of somatic mutations. Notably, 33% (7/21) of LCH cases with wild-type BRAF and none (0/20) with BRAFV600E harbored somatic mutations in MAP2K1 (6 in-frame deletions and 1 missense mutation) that induced extracellular signal-regulated kinase (ERK) phosphorylation in vitro. Single cases of somatic mutations of the mitogen-activated protein kinase (MAPK) pathway genes ARAF and ERBB3 were also detected. The ability of MAPK pathway inhibitors to suppress MAPK kinase and ERK phosphorylation in cell culture and primary tumor models was dependent on the specific LCH mutation. The findings of this study support a model in which ERK activation is a universal end point in LCH arising from pathological activation of upstream signaling proteins.

Mutational Landscape of Aggressive Cutaneous Squamous Cell Carcinoma

Purpose: Aggressive cutaneous squamous cell carcinoma (cSCC) is often a disfiguring and lethal disease. Very little is currently known about the mutations that drive aggressive cSCC. Experimental Design: Whole-exome sequencing was performed on 39 cases of aggressive cSCC to identify driver genes and novel therapeutic targets. Significantly, mutated genes were identified with MutSig or complementary methods developed to specifically identify candidate tumor suppressors based upon their inactivating mutation bias. Results: Despite the very high-mutational background caused by UV exposure, 23 candidate drivers were identified, including the well-known cancer-associated genes TP53, CDKN2A, NOTCH1, AJUBA, HRAS, CASP8, FAT1, and KMT2C (MLL3). Three novel candidate tumor suppressors with putative links to cancer or differentiation, NOTCH2, PARD3, and RASA1, were also identified as possible drivers in cSCC. KMT2C mutations were associated with poor outcome and increased bone invasion. Conclusions: The mutational spectrum of cSCC is similar to that of head and neck squamous cell carcinoma and dominated by tumor-suppressor genes. These results improve the foundation for understanding this disease and should aid in identifying and treating aggressive cSCC. Clin Cancer Res; 20(24); 6582–92. ©2014 AACR.

Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication

Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.

Genomic profiling of Sézary syndrome identifies alterations of key T cell signaling and differentiation genes

Sézary syndrome is a rare leukemic form of cutaneous T cell lymphoma characterized by generalized redness, scaling, itching and increased numbers of circulating atypical T lymphocytes. It is rarely curable, with poor prognosis. Here we present a multiplatform genomic analysis of 37 patients with Sézary syndrome that implicates dysregulation of cell cycle checkpoint and T cell signaling. Frequent somatic alterations were identified in TP53, CARD11, CCR4, PLCG1, CDKN2A, ARID1A, RPS6KA1 and ZEB1. Activating CCR4 and CARD11 mutations were detected in nearly one-third of patients. ZEB1, encoding a transcription repressor essential for T cell differentiation, was deleted in over one-half of patients. IL32 and IL2RG were overexpressed in nearly all cases. Our results demonstrate profound disruption of key signaling pathways in Sézary syndrome and suggest potential targets for new therapies.

Multilevel Genomics-Based Taxonomy of Renal Cell Carcinoma

On the basis of multidimensional and comprehensive molecular characterization (including DNA methalylation and copy number, RNA, and protein expression), we classified 894 renal cell carcinomas (RCCs) of various histologic types into nine major genomic subtypes. Site of origin within the nephron was one major determinant in the classification, reflecting differences among clear cell, chromophobe, and papillary RCC. Widespread molecular changes associated with TFE3 gene fusion or chromatin modifier genes were present within a specific subtype and spanned multiple subtypes. Differences in patient survival and in alteration of specific pathways (including hypoxia, metabolism, MAP kinase, NRF2-ARE, Hippo, immune checkpoint, and PI3K/AKT/mTOR) could further distinguish the subtypes. Immune checkpoint markers and molecular signatures of T cell infiltrates were both highest in the subtype associated with aggressive clear cell RCC. Differences between the genomic subtypes suggest that therapeutic strategies could be tailored to each RCC disease subset.

Integrative Genomic Analysis of Cholangiocarcinoma Identifies Distinct IDH-Mutant Molecular Profiles

Summary Cholangiocarcinoma (CCA) is an aggressive malignancy of the bile ducts, with poor prognosis and limited treatment options. Here, we describe the integrated analysis of somatic mutations, RNA expression, copy number, and DNA methylation by The Cancer Genome Atlas of a set of predominantly intrahepatic CCA cases and propose a molecular classification scheme. We identified an IDH mutant-enriched subtype with distinct molecular features including low expression of chromatin modifiers, elevated expression of mitochondrial genes, and increased mitochondrial DNA copy number. Leveraging the multi-platform data, we observed that ARID1A exhibited DNA hypermethylation and decreased expression in the IDH mutant subtype. More broadly, we found that IDH mutations are associated with an expanded histological spectrum of liver tumors with molecular features that stratify with CCA. Our studies reveal insights into the molecular pathogenesis and heterogeneity of cholangiocarcinoma and provide classification information of potential therapeutic significance.

Loss of Rho GDIα and Resistance to Tamoxifen via Effects on Estrogen Receptor α

BACKGROUND Estrogen receptor (ER) α is a successful therapeutic target in breast cancer, but patients eventually develop resistance to antiestrogens such as tamoxifen. METHODS To identify genes whose expression was associated with the development of tamoxifen resistance and metastasis, we used microarrays to compare gene expression in four primary tumors from tamoxifen-treated patients whose breast cancers did not recur vs five metastatic tumors from patients whose cancers progressed during adjuvant tamoxifen treatment. Because Rho guanine dissociation inhibitor (GDI) α was underexpressed in the tamoxifen-resistant group, we stably transfected ERα-positive MCF-7 breast cancer cells with a plasmid encoding a short hairpin (sh) RNA to silence Rho GDIα expression. We used immunoblots and transcription assays to examine the role of Rho GDIα in ER-related signaling and growth of cells in vitro and as xenografts in treated nude mice (n = 8-9 per group) to examine the effects of Rho GDIα blockade on hormone responsiveness and metastatic behavior. The time to tumor tripling as the time in weeks from randomization to a threefold increase in total tumor volume over baseline was examined in treated mice. The associations of Rho GDIα and MTA2 levels with tamoxifen resistance were examined in microarray data from patients. All statistical tests were two-sided. RESULTS Rho GDIα was expressed at lower levels in ERα-positive tumors that recurred during tamoxifen treatment than in ERα-positive tamoxifen-sensitive primary tumors. MCF-7 breast cancer cells in which Rho GDIα expression had been silenced were tamoxifen-resistant, had increased Rho GTPase and p21-activated kinase 1 activity, increased phosphorylation of ERα at serine 305, and enhanced tamoxifen-induced ERα transcriptional activity compared with control cells. MCF-7 cells in which Rho GDIα expression was silenced metastasized with high frequency when grown as tumor xenografts. When mice were treated with estrogen or estrogen withdrawal, tripling times for xenografts from cells with Rho GDIα silencing were similar to those from vector-containing control cells; however, tripling times were statistically significantly faster than control when mice were treated with tamoxifen (median tripling time for tumors with Rho GDIα small interfering RNA = 2.34 weeks; for control tumors = not reached, hazard ratio = 4.13, 95% confidence interval = 1.07 to 15.96, P = .040 [adjusted for multiple comparisons, P = .119]). Levels of the metastasis-associated protein MTA2 were also increased upon Rho GDIα silencing, and combined Rho GDIα and MTA2 levels were associated with recurrence in 250 tamoxifen-treated patients. CONCLUSION Loss of Rho GDIα enhances metastasis and resistance to tamoxifen via effects on both ERα and MTA2 in models of ERα-positive breast cancer and in tumors of tamoxifen-treated patients.

Dicer-Mediated Upregulation of BCRP Confers Tamoxifen Resistance in Human Breast Cancer Cells

Purpose: Tamoxifen (Tam) is the most prescribed hormonal agent for treatment of estrogen receptor α (ERα)-positive breast cancer patients. Using microarray analysis, we observed that metastatic breast tumors resistant to Tam therapy had elevated levels of Dicer. Experimental Design: We overexpressed Dicer in ERα-positive MCF-7 human breast cancer cells and observed a concomitant increase in expression of the breast cancer resistance protein (BCRP). We thus hypothesized that Tam resistance associated with Dicer overexpression in ERα-positive breast cancer cells may involve BCRP. We analyzed BCRP function in Dicer-overexpressing cells using growth in soft agar and mammosphere formation and evaluated intracellular Tam efflux. Results: In the presence of Tam, Dicer-overexpressing cells formed resistant colonies in soft agar, and treatment with BCRP inhibitors restored Tam sensitivity. Tumor xenograft studies confirmed that Dicer-overexpressing cells were resistant to Tam in vivo. Tumors and distant metastases could be initiated with as few as five mammosphere cells from both vector and Dicer-overexpressing cells, indicating that the mammosphere assay selected for cells with enhanced tumor-initiating and metastatic capacity. Dicer-overexpressing cells with elevated levels of BCRP effluxed Tam more efficiently than control cells, and BCRP inhibitors were able to inhibit efflux. Conclusion: Dicer-overexpressing breast cancer cells enriched for cells with enhanced BCRP function. We hypothesize that it is this population which may be involved in the emergence of Tam-resistant growth. BCRP may be a novel clinical target to restore Tam sensitivity. Clin Cancer Res; 17(20); 6510–21. ©2011 AACR.