Lisa R. Trevino

Exome Sequencing of Head and Neck Squamous Cell Carcinoma Reveals Inactivating Mutations in NOTCH1

The mutational profile of head and neck cancer is complex and may pose challenges to the development of targeted therapies. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. To explore the genetic origins of this cancer, we used whole-exome sequencing and gene copy number analyses to study 32 primary tumors. Tumors from patients with a history of tobacco use had more mutations than did tumors from patients who did not use tobacco, and tumors that were negative for human papillomavirus (HPV) had more mutations than did HPV-positive tumors. Six of the genes that were mutated in multiple tumors were assessed in up to 88 additional HNSCCs. In addition to previously described mutations in TP53, CDKN2A, PIK3CA, and HRAS, we identified mutations in FBXW7 and NOTCH1. Nearly 40% of the 28 mutations identified in NOTCH1 were predicted to truncate the gene product, suggesting that NOTCH1 may function as a tumor suppressor gene rather than an oncogene in this tumor type.

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.

Identification of TP53 as an acute lymphocytic leukemia susceptibility gene through exome sequencing.

Although acute lymphocytic leukemia (ALL) is the most common childhood cancer, genetic predisposition to ALL remains poorly understood. Whole‐exome sequencing was performed in an extended kindred in which five individuals had been diagnosed with leukemia. Analysis revealed a nonsense variant of TP53 which has been previously reported in families with sarcomas and other typical Li Fraumeni syndrome‐associated cancers but never in a familial leukemia kindred. This unexpected finding enabled identification of an appropriate sibling bone marrow donor and illustrates that exome sequencing will reveal atypical clinical presentations of even well‐studied genes. Pediatr Blood Cancer 2013; 60: E1–E3.

Genomic profiling of pediatric acute myeloid leukemia reveals a changing mutational landscape from disease diagnosis to relapse

The genomic and clinical information used to develop and implement therapeutic approaches for acute myelogenous leukemia (AML) originated primarily from adult patients and has been generalized to patients with pediatric AML. However, age-specific molecular alterations are becoming more evident and may signify the need to age-stratify treatment regimens. The NCI/COG TARGET-AML initiative used whole exome capture sequencing (WXS) to interrogate the genomic landscape of matched trios representing specimens collected upon diagnosis, remission, and relapse from 20 cases of de novo childhood AML. One hundred forty-five somatic variants at diagnosis (median 6 mutations/patient) and 149 variants at relapse (median 6.5 mutations) were identified and verified by orthogonal methodologies. Recurrent somatic variants [in (greater than or equal to) 2 patients] were identified for 10 genes (FLT3, NRAS, PTPN11, WT1, TET2, DHX15, DHX30, KIT, ETV6, KRAS), with variable persistence at relapse. The variant allele fraction (VAF), used to measure the prevalence of somatic mutations, varied widely at diagnosis. Mutations that persisted from diagnosis to relapse had a significantly higher diagnostic VAF compared with those that resolved at relapse (median VAF 0.43 vs. 0.24, P < 0.001). Further analysis revealed that 90% of the diagnostic variants with VAF >0.4 persisted to relapse compared with 28% with VAF <0.2 (P < 0.001). This study demonstrates significant variability in the mutational profile and clonal evolution of pediatric AML from diagnosis to relapse. Furthermore, mutations with high VAF at diagnosis, representing variants shared across a leukemic clonal structure, may constrain the genomic landscape at relapse and help to define key pathways for therapeutic targeting. Cancer Res; 76(8); 2197-205. ©2016 AACR.

Genomic analysis of hepatoblastoma identifies distinct molecular and prognostic subgroups

Despite being the most common liver cancer in children, hepatoblastoma (HB) is a rare neoplasm. Consequently, few pretreatment tumors have been molecularly profiled, and there are no validated prognostic or therapeutic biomarkers for HB patients. We report on the first large‐scale effort to profile pretreatment HBs at diagnosis. Our analysis of 88 clinically annotated HBs revealed three risk‐stratifying molecular subtypes that are characterized by differential activation of hepatic progenitor cell markers and metabolic pathways: high‐risk tumors were characterized by up‐regulated nuclear factor, erythroid 2–like 2 activity; high lin‐28 homolog B, high mobility group AT‐hook 2, spalt‐like transcription factor 4, and alpha‐fetoprotein expression; and high coordinated expression of oncofetal proteins and stem‐cell markers, while low‐risk tumors had low lin‐28 homolog B and lethal‐7 expression and high hepatic nuclear factor 1 alpha activity. Conclusion: Analysis of immunohistochemical assays using antibodies targeting these genes in a prospective study of 35 HBs suggested that these candidate biomarkers have the potential to improve risk stratification and guide treatment decisions for HB patients at diagnosis; our results pave the way for clinical collaborative studies to validate candidate biomarkers and test their potential to improve outcome for HB patients. (Hepatology 2017;65:104‐121).

Abstract 4267: Integrated genomic analysis of hepatoblastoma identifies distinct molecular and prognostic subgroups

Hepatoblastoma (HB), the most common primary liver cancer in children, is a clinically heterogeneous embryonal malignancy. Previous studies of HB have revealed a paucity of genetic alterations in these tumors, other than mutations activating the WNT signaling pathway, and have not provided an adequate biological explanation for the diverse clinical outcomes of children with HB. To identify critical genes and pathways in the pathogenesis of HB and provide insight into the biologic basis and clinical heterogeneity of these tumors, we performed an integrated genomic analysis of a large cohort of clinically-annotated tumor-normal pairs utilizing whole exome sequencing (n = 35), mRNA and miRNA expression arrays (n = 51; n = 58), and high-resolution copy number arrays (n = 47). Somatic mutations/deletions of CTNNB1 or rare germline mutations of APC were identified in >90% of cases, confirming the central role of the WNT pathway in HB pathogenesis. The only other gene targeted by recurrent mutations in our cohort was the transcription factor NFE2L2 (NRF2), which was found to harbor hotspot mutations at a similar frequency (5-10%) to that reported for hepatocellular carcinoma. Expression profiling revealed near-universal WNT-pathway activation: 7 of the 20 most highly expressed genes in HB were WNT-pathway related genes, and the WNT-pathway marker DKK1 was expressed hundreds of standard deviations above its levels in normal liver. Unsupervised hierarchical clustering identified three distinct molecular HB clusters that were characterized by differential activation of hepatic progenitor cell and metabolic pathways. Analysis of the expression and/or inferred-activity of transcription factors and highly expressed genes identified prognostic biomarkers, including the significantly predictive regulons of NFE2L2, LIN28b, HNF1A, and NOTCH1. These were used to identify clinical groups that overlapped with HB expression clusters and were associated with patient survival. Tumors in the high-risk group were characterized by high NFE2L2, low NOTCH1 activity, and high LIN28b expression and activity (corresponding to low let-7b expression), as well as high expression of the onco-fetal proteins AFP and GPC3 and stem cell markers EPCAM, DLK1, and SALL4. In contrast, the low-risk group was characterized by high HNF1a and NOTCH1 activities and low LIN28 expression and activity. Rare chromosomal gains and losses were characteristic of the low risk group of tumors, while gains of 20q13 (SALL4) and 1q chromosomal regions were primarily found in the high and intermediate risk groups. Immunohistochemistry for LIN28b, NFE2L2, HNF1alpha, PTEN, and EPCAM is being performed on a validation set of tumors to assess their reliability to classify formalin-fixed tumor specimens. Prospective studies will test the prognostic utility of this method for children with HB. Citation Format: Dolores H. Lopez-Terrada, Pavel Sumazin, Yidong Chen, Lisa Trevino, Stephen Sarabia, Oliver Hampton, Kayuri Patel, Toni-Ann Mistretta, Barry Zorman, Sarah Comerford, David Wheeler, Murali Chintagumpala, Rebecka M. Meyers, Milton J. Finegold, Gail Tomlinson, Donald W. Parsons. Integrated genomic analysis of hepatoblastoma identifies distinct molecular and prognostic subgroups. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4267. doi:10.1158/1538-7445.AM2015-4267

Abstract A17: Silencing of Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) impairs growth and proliferation of hepatoblastoma cells

Background: Hepatoblastoma (HB) is the most common primary, malignant liver tumor in children, occurring primarily in children under the age of 4 years. Although chemotherapy in conjunction with surgical resection has improved the prognosis for patients with HB, disease-free survival rates remain less than 50% for patients with advanced, metastatic or relapsed disease, highlighting the need for novel treatments. Additional subsets of tumors with a worse prognosis include those with histologic features of hepatocellular carcinoma (HCC) and tumors which develop in children older than 4 years. Nuclear factor erythroid-2–related factor 2 (NFE2L2, also known as Nrf2), a key transcriptional regulator for the cellular response to oxidative stress in normal cells, has been shown to contribute to malignant phenotypes of cancers. Recently, activating mutations in NFE2L2 have been reported in HCC and also in HB by our research team (Trevino et al 1 ). These activating mutations of NFE2L2 and/or high expression of NFE2L2 have been observed notably in several pediatric liver tumors with high-risk features associated with a poor outcome. We hypothesize that NFE2L2 activation/overexpression may contribute to an aggressive tumor phenotype, and investigated its role in HB cell proliferation. Methods: Hep293TT cells, previously established using primary tumor tissues from a highly aggressive hepatoblastoma from a 5-year-old Caucasian female, were transfected with NFE2L2 small hairpin RNAs (shRNAs) to determine the effect of NFE2L2 suppression on cell proliferation and to identify potential NFE2L2-regulated targets. Analyses of Hep293TT cell proliferation and cell cycle kinetics were conducted using cell viability assays (CellTiter-Glo Luminescence), propidium iodide staining and FACS analysis. A comprehensive transcriptome analyses of Hep293TT cells was carried out by performing RNA sequencing (RNA-seq) using an Illumina HiSeq 2000. Results: RNA-seq analysis was performed on a set of pediatric cancer cell lines including Hep293TT, as well as reference liver cell lines HepG2 and Hep3B. After normalization, the analysis revealed high expression of NFE2L2 in Hep293TT cells with fold change of 2.01 relative to the mean expression level from the pediatric cancer cell line panel (14 cell lines). Compared to other liver cancer cell lines, fold change of 1.28 and 1.5 were measured from Hep3B and HepG2, respectively. Differential expression between NFE2L2 knockdown and parental cell will further demonstrate the molecular and functional influence of NFE2L2. Furthermore, knockdown of NFE2L2 inhibited cell proliferation in Hep293TT cells by inducing cycle arrest. Conclusions: Our results suggest that NFE2L2 may contribute to cell growth and proliferation in hepatoblastoma. Further studies are warranted to determine if NFE2L2 status can be used to stratify highly aggressive pediatric liver tumors and establish NFE2L2 as a potentially novel target for the treatment of HBs. 1 Trevino et al, AACR Annual Meeting, 2012, Abstract #4592 This work was supported by grants from the Cancer Prevention and Research Institute of Texas RP101195 and NIH R21 CA122565. Citation Format: Hima Bansal, Tina Chen, Zhao Lai, Donald Parsons, Lisa Trevino, Dolores Lopez-Terrada, Milton Finegold, Dinesh Rakheja, Sarah Comerford, Robert Hammer, Bradley Pollock, Jaclyn Hung, Sanjay Bansal, Yidong Chen, Gail Tomlinson. Silencing of Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) impairs growth and proliferation of hepatoblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A17.

Abstract 4592: Exome sequencing of hepatoblastoma reveals recurrent mutations in NFE2L2.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Hepatoblastoma (HB), the most common malignant liver tumor in children, is a biologically and clinically heterogeneous embryonal malignancy. Activation of the Wnt pathway is known to occur in the vast majority of HBs, most frequently through somatic mutation of CTNNB1 and less often from germline mutation of APC, but knowledge of other gene alterations is scant. To identify critical genes and pathways in the pathogenesis of HB and provide insight into potential clinically-relevant targets, we performed whole exome sequencing on 35 clinically-annotated tumor-normal pairs. Putative mutations identified through exome sequencing on Illumina-based instruments were confirmed on a second sequencing platform. A total of 130 somatic mutations were identified in 24 patients (3.7 mutations per tumor; range of 0 to 22 mutations), resulting in a somatic mutation rate of < 0.2 mutations per Mb. CTNNB1 mutations were identified in 13 HBs (37%), confirming the central role of the Wnt pathway in HB. Somatic mutations were also found in other cancer genes, including the chromatin-remodeling genes MLL2 and ARID1A. Frequent somatic mutations were identified in genes related to regulation of oxidative stress, including recurrent point mutations in NFE2L2 (NRF2) and inactivating mutations in the thioredoxin-domain containing genes TXNDC15 and TXNDC16. An additional 27 HBs were sequenced for NFE2L2 mutations, revealing a mutation frequency of 6.5% (4 of 62 tumors). Mutations of NFE2L2 are known to occur in 5-10% of adult hepatocellular carcinomas (HCC) as well as in other tumor types: as in those tumors, the NFE2L2 mutations identified in HB (p.D29N in one patient and p.R34G in three patients) are clustered within the Neh2 domain and are expected to inhibit KEAP1-mediated degradation of NRF2, resulting in stabilization and nuclear accumulation of NRF2 and activation of downstream oxidative stress response genes. Most NFE2L2-mutated adult HCCs also contain mutations in CTNNB1 or other Wnt pathway genes, suggesting a biological link between NFE2L2-mutated HB and HCC. In addition, whole exome and SNP array data (Affymetrix 6.0 SNPChip) have revealed copy number alterations in previously-described regions of the HB genome, including 1q, 4q and 11p15, as well as novel focal alterations. Finally, germline variants in APC and other Wnt pathway genes have also been identified in this HB patient cohort. In summary, next-generation sequencing of HB has provided an unprecedented view of the genetic landscape of HB, confirmed the primary importance of dysregulation of Wnt signaling in this tumor type, and revealed recurrent hotspot mutations in NFE2L2, a potential therapeutic target. Supported by the Cancer Prevention & Research Institute of Texas (RP101195) and the National Institutes of Health (CA098543). Citation Format: Lisa R. Trevino, David A. Wheeler, Milton J. Finegold, Murali Chintagumpala, Kayuri U. Patel, Stephen F. Sarabia, Sarah A. Comerford, Robert E. Hammer, Dinesh Rakheja, Rebecka L. Meyers, Yidong Chen, Bradley H. Pollock, Gail E. Tomlinson, Dolores H. Lopez-Terrada, D. Williams Parsons. Exome sequencing of hepatoblastoma reveals recurrent mutations in NFE2L2 . [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 4592. doi:10.1158/1538-7445.AM2013-4592

Abstract 5113: Functional analysis of genomic variants identified through whole exome sequencing for susceptibility to lymphocytic leukemia

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The underlying genetic basis of many childhood cancers remains largely unknown, which places a significant focus on the discovery and understanding of cancer susceptibility genes. Our research is focused on the identification of novel high risk childhood cancer susceptibility genes through next generation sequencing methods and functional assays. One kindred contained four individuals diagnosed during childhood with lymphocytic leukemia or lymphoma transmitted in an autosomal dominant pattern of inheritance. Whole exome sequencing was performed on the constitutional DNA of three of the patients diagnosed with childhood cancer and one non-transmitting parent as an internal control. This analysis resulted in over 1000 unique single nucleotide variant (SNV) sites shared among the three affected family members. Of these SNVs, greater than 95% were found in control databases and excluded. Also, any variant found in the non-transmitting parent was eliminated. Systematic bioinformatics analysis resulted in a short list of seven missense SNVs predicted to be functionally important. Within this list is the missense mutation of a conserved residue (L254P) of Human Cytosolic 5′ Nucleotidase (NT5C1A). NT5C1A has a role in nucleoside metabolism as this protein primarily catalyzes the production of adenosine through the dephosphorylation of AMP. Adenosine metabolism is essential for lymphocyte maturation and viability, making NT5C1A an attractive leukemia susceptibility candidate to pursue further through functional genomic assays. Prior studies have shown that overexpression of wild type NT5C1A in HEK293 cells improves cell survival after treatment with purine and pyrimidine analogs also used in leukemia treatment. We created multiple HEK293 cell lines that stably overexpress either the NT5C1A wild type or L254P mutant protein. These cells were tested for quantitative changes in NT5C1A mediated cytotoxicity to the nucleoside analogs, Cladribine and Gemcitabine. We have replicated the prior findings in cells overexpressing wild type NT5C1A and demonstrated that resistance is dependent on the amount of exogenous NT5C1A protein expressed. Importantly, overexpression of the L254P protein is deficient in mediating resistance to these analogs, which is consistent with this novel variant interfering with NT5C1A activity. This finding has led us to hypothesize that the L254P mutation leads to a reduction in protein function, most likely through reduced substrate binding, which may increase AMP levels in early lymphocytes and predispose to tumorigenesis. Future directions include experiments to determine the L254P proteins substrate binding affinity and activity and the introduction of the mutation into murine leukemia models as well as analysis of L254P as a pharmacogenetic variant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5113. doi:1538-7445.AM2012-5113