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Annals of diagnostic pathology  2013;18(1):10-13.
Notch signaling plays role in stem cell biology, tumor formation, angiogenesis, and cell death. Targeting Notch pathway could serve as a therapeutic strategy in cancer. Little is known about the differential role of various components of the Notch pathway in salivary AdCC.
To investigate the association of the Notch pathway in AdCC carcinogenesis, we analyzed the Notch receptors (Notch-1, Notch-2, Notch-4) and Notch ligands (Jagged-1, Delta) expressions.
The results showed elevated expression levels of all five proteins in AdCC tissue relative to normal salivary gland tissues. Jagged-1/Notch-2 co-expression was significantly associated with increased patient survival rate. The elevated expression level of these Notch receptors and ligands in AdCC points to Notch signaling as a key player in AdCC pathogenesis.
Our data provide evidence for relationship between Jagged-1/Notch-2 co-expression and better overall patient survival with AdCC. Targeting Notch signaling pathway may provide therapeutic benefits for these patients.
PMCID: PMC4807115  PMID: 24238845
adenoid cystic; carcinoma; Notch; cancer stem cells; targeted therapy; Notch-2/ Jagged-1
2.  Alterations Associated with Androgen Receptor Gene Activation in Salivary Duct Carcinoma (SDC) of Both Sexes: Potential Therapeutic Ramifications 
To investigate the molecular events associated with the activation of androgen receptor (AR) as a potential therapeutic target in patients with salivary duct carcinoma (SDC).
Experimental Design
Comprehensive molecular and expression analysis of the AR gene in 35 tumor specimens (20 males and 15 females) and cell lines derived from SDC using Western blotting and RT-PCR, FISH analysis, and DNA sequencing were conducted. In vitro and in vivo animal studies were also performed.
AR expression was detected in 70% of the tumors and was mainly nuclear and homogenous in both male and female SDCs, although variable cytoplasmic and/or nuclear localization was also found. We report the identification of Ligand-independent AR splice variants, mutations and extra AR gene copy in primary untreated SDC tumors. In contrast to prostate cancer, no AR gene amplification was observed. In vitro knockdown of AR in a female derived SDC cell line revealed marked growth inhibition in culture and in vivo androgen independent tumor growth.
Our study provides new detailed information on the molecular and structural alterations associated with AR gene activation in SDC and shed more light on the putative functional role of AR in SDC cells. Based on these data, we propose that patients with SDC (male and female) can be stratified for hormone-based therapy in future clinical trials.
PMCID: PMC4268116  PMID: 25316813
Salivary duct carcinoma; Androgen receptor; Splice variants; Copy number alterations; Androgen Resistance
3.  Molecular and Phenotypic Analysis of Poorly Differentiated Sinonasal Neoplasms: An Integrated Approach for Early Diagnosis and Classification 
Human pathology  2009;40(3):283-292.
Accurate early diagnosis of poorly differentiated tumors of the sinonasal and skull-base sites is critical to modern multimodality management of patients with these tumors. A combined phenotypic and sequential biomarkers approach of a large retrospective cohort of these tumors led to the reclassification of some cases and the confirmation of uncertain diagnoses in others. An integrated algorithm of selected markers and phenotypic features for biopsy-based diagnosis of these tumors is presented and discussed.
Primary poorly differentiated (small round and non-small) sinonasal neoplasms comprise histogenetically and biologically diverse entities with overlapping morphologic features. Because of the limited initial biopsy tissue materials, differential diagnostic difficulties may arise and complicate timely management of some cases. We employed immunohistochemical and molecular marker analyses in a large cohort of these tumors to optimize their early diagnosis and classification. Fifty-two tumors of the skull base and sinonasal regions and, for comparison, 19 poorly differentiated neoplasms of other head and neck sites were analyzed by a panel of immunohistochemical markers including those of epithelial, mesenchymal, melanocytic, and neuroectodermal origin using tissue microarray. RT-PCR analysis of mRNA for EWS-FLI1 and PAX-FKHR fusion transcripts and the hASH1 gene was performed on 24 of the 52 sinonasal tumors and the 19 tumors of other sites for comparison. The immunohistochemical results substantiated the phenotypic assessment and the initial diagnosis in 49 of the 52 tumors. In four instances the integrated markers and phenotypic analyses led to reclassification of three tumors and confirmed the histogenesis of a mesenchymal tumor with aberrant cytokeratin expression. Molecular analysis of the EWS-FLI1 fusion gene transcript revealed four (9.3%) of the 43 tumors to be positive; all were Ewing’s sarcomas. The hASH1 gene transcript was identified in 10 (23.8%) of 42 tumors: three of six neuroblastomas, all four neuroendocrine carcinomas, and one each in sinonasal undifferentiated carcinoma, rhabdomyosarcoma, and melanoma. The PAX-FKHR fusion transcript was not detected in any tumors. We conclude that 1) an integrated morphologic and biomarker algorithm may better optimize the early diagnosis of poorly differentiated sinonasal and skull-base tumors; 2) molecular analysis may assist in future biological stratification of certain classes of these tumors; and 3) the hASH1 gene transcript is a nonspecific marker for the diagnosis of neuroblastoma.
PMCID: PMC4573623  PMID: 19150107
Sinonasal tumors; Poorly differentiated; Diagnostic profiling; ‘small round cell’ neoplasms; Molecular classification
4.  Genetic and Expression Analysis of HER-2 and EGFR Genes in Salivary Duct Carcinoma: Empirical and Therapeutic Significance 
Salivary duct carcinoma over-expresses epidermal growth factor receptor (EGFR) and HER-2 though underlying mechanisms remain undefined. Because of potential utilization of these markers as treatment targets, we evaluated protein and gene status by several techniques to determine complementary value.
Experimental Design
A tissue microarray of 66 salivary duct carcinomas was used for immunohistochemical analysis of HER-2 and EGFR expression (semiquanititively evaluated into a 3-tiered system), and fluorescence in situ hybridization for gene copy number, and chromosomes 7 and 17 ploidy status. Sequencing of Exons 18, 19 and 21 of the EGFR gene for mutations was performed.
EGFR Forty-six (69.7%) of the 66 tumors showed some form of EGFR expression (17,3+; 17,2+; 12,1+) but none gene amplification. Five (9.4%) of 53 tumors showed mutations in exon 18 (3) and exon 19 (2). Polysomy of chromosome 7(average >2.5 copies per cell) was detected in 15 (25.0%) of 60 tumors (6,3+; 5,2+; 2,1+; 2,0+ expression) and correlated with poor 3-year survival (p=0.015). HER-2: Seventeen (25.8%) of 66 tumors expressed HER-2 (10,3+; 3,2+; 4,1+). Eight tumors showed HER-2 gene amplification (6,3+; 1,1+; 1,0+ protein expression). Chromosome 17 polysomy was found in eight (15.7%) of 51 tumors; two with HER-2 expression (3+; 1+).
Our study shows that salivary duct carcinomas: 1) harbor EGFR gene mutations in a subset of tumors which may guide therapy, 2) pursue aggressive clinical course in cases with Chromosome 7 polysomy and high EGFR expression, and 3) with HER-2 gene amplification and protein high-expression maybe selected for targeted therapy.
PMCID: PMC4152860  PMID: 20371674
Salivary duct carcinoma; epidermal growth factor receptor; HER-2; Fluorescence in situ hybridization; polysomy; mutation
5.  A Novel Target for Oral Cancer Chemoprevention? Notch Quite, Yet… 
The two major goals of oral cancer chemoprevention efforts are the ability to segregate the high-risk patients and the identification of an effective pharmacologic agent that halts progression to invasive cancer. Considerable progress has recently been achieved in profiling invasive head and neck squamous cell carcinomas, particularly with the use of high throughput technologies. Similar molecular characterization of potentially malignant oral epithelial lesions (OPMLs) [leukoplakia and erythroplakia] is yet to be accomplished. It is postulated, though, that molecular profiling could lead to the discovery of novel markers of cancer risk that could also serve as potential targets for chemoprevention. In this perspective, we comment on the work by Izumchenko et al. that reports a high prevalence of NOTCH1 gain of function mutations in Chinese patients with OPMLs. Although additional studies are needed to validate the findings, the study is the first to link alterations in this gene in oral premalignancy. These findings could serve as a first prototype of a single gene mutation as a potential target in clinical chemoprevention setting.
PMCID: PMC4383667  PMID: 25712052
oral premalignant lesions; leukoplakia; chemoprevention; NOTCH1
6.  Evolutionary Action score of TP53 (EAp53) identifies high risk mutations associated with decreased survival and increased distant metastases in head and neck cancer 
Cancer research  2015;75(7):1527-1536.
TP53 is the most frequently altered gene in head and neck squamous cell carcinoma (HNSCC) with mutations occurring in over two third of cases, but the prognostic significance of these mutations remains elusive. In the current study, we evaluated a novel computational approach termed Evolutionary Action (EAp53) to stratify patients with tumors harboring TP53 mutations as high or low risk, and validated this system in both in vivo and in vitro models. Patients with high risk TP53 mutations had the poorest survival outcomes and the shortest time to the development of distant metastases. Tumor cells expressing high risk TP53 mutations were more invasive and tumorigenic and they exhibited a higher incidence of lung metastases. We also documented an association between the presence of high risk mutations and decreased expression of TP53 target genes, highlighting key cellular pathways that are likely to be dysregulated by this subset of p53 mutations which confer particularly aggressive tumor behavior. Overall, our work validated EAp53 as a novel computational tool that may be useful in clinical prognosis of tumors harboring p53 mutations.
PMCID: PMC4383697  PMID: 25634208
Head and neck squamous cell carcinoma; TP53; prognostic biomarker
7.  Whole exome sequencing of adenoid cystic carcinoma 
The Journal of Clinical Investigation  2013;123(7):2965-2968.
Adenoid cystic carcinoma (ACC) is a rare malignancy that can occur in multiple organ sites and is primarily found in the salivary gland. While the identification of recurrent fusions of the MYB-NFIB genes have begun to shed light on the molecular underpinnings, little else is known about the molecular genetics of this frequently fatal cancer. We have undertaken exome sequencing in a series of 24 ACC to further delineate the genetics of the disease. We identified multiple mutated genes that, combined, implicate chromatin deregulation in half of cases. Further, mutations were identified in known cancer genes, including PIK3CA, ATM, CDKN2A, SF3B1, SUFU, TSC1, and CYLD. Mutations in NOTCH1/2 were identified in 3 cases, and we identify the negative NOTCH signaling regulator, SPEN, as a new cancer gene in ACC with mutations in 5 cases. Finally, the identification of 3 likely activating mutations in the tyrosine kinase receptor FGFR2, analogous to those reported in ovarian and endometrial carcinoma, point to potential therapeutic avenues for a subset of cases.
PMCID: PMC3999050  PMID: 23778141
8.  Clinical significance of Myb protein and downstream target genes in salivary adenoid cystic carcinoma 
Cancer Biology & Therapy  2011;12(7):569-573.
Adenoid cystic carcinoma (ACC), the second most frequent malignancy of the major and minor salivary glands, comprise of approximately 15–23% of all carcinomas at these locations. ACC is uniquely formed of dual epithelial and myoepithelial cells that give rise to different phenotypic patterns. We hypothesize that the dual myoepithelial/epithelial composition of ACCs underlie their biological heterogeneity and may impact on their therapeutic management. A recurrent reciprocal translocation of t(6;9)(q22–23;p23–24) resulting in fusion gene partners comprising MYB gene the transcription factor NFIB has been reported in ACC of breast, salivary, lachrymal and ceruminal glands. In fusion positive and a subset of fusion negative ACCs, high expression of the transcript Myb was found. However, the role of Myb protein expression and the potential effect on the downstream targets have not been investigated. To investigate the biological and prognostic significance of use of elevated levels of Myb and its downstream target genes (c-kit, cox-2, bcl-2), we analyzed, by immunohistochemistry, the protein expression of these genes in 156 ACCs.
We have found that 55% of ACCs have increased Myb expression mainly confined to myoepithelial cells. We validated Myb expression on a large cohort of ACCs (156 patients). Although no significant effects of the individual Myb and downstream targets c-kit, bcl-2 and cox-2 on survival was noticed, the combinations survival curve for Myb+/c-kit+/cox-2+ showed better survival than combination Myb−/c-kit+/cox-2+. Myb may serve as a new target for the management of this disease, and future therapeutic trials of these tumors may be better based on biomarker stratification and the cellular composition of these tumors.
PMCID: PMC3218383  PMID: 21785271
adenoid cystic carcinoma; c-kit; cox-2; bcl-2
9.  Targeting the Mitogen-Activated Protein Kinase RAS-RAF Signaling Pathway in Cancer Therapy 
The mitogen-activated protein kinase (MAPK) pathway comprises several key signaling components and phosphorylation events that play important role in tumorigenesis. These activated kinases transmit extracellular signals that regulate cell growth, differentiation, proliferation, apoptosis and migration functions. Alteration of the RAS-RAF-MEK-ERK-MAPK (RAS-MAPK) pathway has frequently been reported in human cancer as a result of abnormal activation of receptor tyrosine kinases or gain-of-function mutations mainly in the RAS or RAF genes. Accordingly, these pathways are considered a potential therapeutic target for cancer treatment. Recently, several small-molecule inhibitors targeting this pathway have been developed and are currently being tested in clinical trials.
Areas covered
This paper focuses on the biological role of the RAS-MAPK pathway, the consequence of its disregulation, and the development of small-molecule inhibitors. The rationale for targeting the RAS-MAPK pathway will be reviewed here along with a discussion of the application and the results of various inhibitory molecules as anticancer agents in clinical trials.
Expert opinion
The RAS-MAPK pathway mediates cellular responses to growth signals and is often deregulated in human cancer. Activating mutations in the RAS and BRAF genes have been frequently identified in a wide range of cancers. Inhibitors of MEK and particularly of RAF kinases, have been effective in clinical trials with manageable side effects. RAS and BRAF genes need to be analyzed for mutations as markers of response to treatments and to avoid paradoxical effects. Further characterization of the RAS-MAPK molecular mechanisms regulation in malignant cells or underlying the acquired resistance to RAF inhibitors will facilitate development of novel combination therapies.
PMCID: PMC3457779  PMID: 22239440
mitogen-activated protein kinase (MAPK); extracellular signal-regulated kinase (ERK); MAP kinase kinase (MEK); RAS; RAF; inhibitors; targeted therapies
10.  Differential gene expression profiling of aggressive and non-aggressive follicular carcinomas 
Human pathology  2011;42(9):1213-1220.
The classification of follicular thyroid neoplasms requires surgical resection for histologic evaluation of malignancy. As variable clinical behavior exists, genomic expression profiling may lead to the identification of novel markers that facilitate better biological classification. We performed for the first time gene expression analysis on clinically aggressive and non-aggressive follicular carcinomas (FCs) from patients for whom long-term follow-up data were available. We examined matched fresh/frozen tissue from 15 histopathologically diagnosed FCs (7 patients with documented distant metastasis and/or death from disease and 8 patients without recurrence). For categorical comparison, we analyzed 4 follicular adenomas (FAs). The biological control comprised 11 normal thyroid tissue specimens. High-quality RNA was extracted from the tissues, labeled, and hybridized to an Affymetrix oligonucleotide microarray (HG-U133A). With the exceptions of 1 FA and 1 FC, unsupervised hierarchical cluster analysis revealed 2 distinct groups—one containing normal thyroid tissue and FAs and another containing FCs. We identified 421 genes that were differentially expressed between histologically normal thyroid tissues and all follicular neoplasms (P < 0.01; fold-change >2), 94 genes that distinguished FCs from FAs (including PBP and CKS2), and 4 genes that distinguished aggressive FCs from non-aggressive FCs (NID2, TM7SF2, TRIM2 and GLTSCR2). Comparative genomic groupings identified differentially expressed genes that may lead to better classification of follicular thyroid neoplasms. Such genes may be used in future prospective validation studies to establish clinically useful and complementary diagnostic markers.
PMCID: PMC3156288  PMID: 21420716
Follicular neoplasms; follicular carcinoma; gene expression; Affymetrix
11.  Novel Chromosomal Rearrangements and breakpoints at the t(6;9) in Salivary Adenoid Cystic Carcinoma: association with MYB-NFIB chimeric fusion, MYB expression, and clinical outcome 
To investigate the molecular-genetic heterogeneity associated with the t(6:9) in adenoid cystic carcinoma (ACC) and correlate the findings with patient clinical outcome.
Experimental Design
Multi-molecular and genetic techniques complemented with massive pair-ended sequencing and SNP array analyses were used on tumor specimens from 30 new and 52 previously RT-PCR analyzed fusion transcript negative ACCs. MYB mRNA expression level was determined by quantitative RT-PCR. The results of 102 tumors (30 new and 72 previously reported cases) were correlated with the clinicopathologic factors and patients’ survival.
The FISH analysis showed 34/82 (41.5%) fusion positive tumors and molecular techniques identified fusion transcripts in 21 of the 82 (25.6%) tumors. Detailed FISH analysis of 11 out the 15 tumors with gene fusion without transcript formation showed translocation of NFIB sequences to proximal or distal sites of the MYB gene. Massive pair-end sequencing of a subset of tumors confirmed the proximal translocation to an NFIB sequence and led to the identification of a new fusion gene (NFIB-AIG1) in one of the tumors. Overall, MYB-NFIB gene fusion rate by FISH was in 52.9% while fusion transcript forming incidence was 38.2%. Significant statistical association between the 5′ MYB transcript expression and patient survival was found.
We conclude that: 1) t(6;9) results in a complex genetic and molecular alterations in ACC, 2) MYB-NFIB gene fusion may not always be associated with chimeric transcript formation, 3) non-canonical MYB, NFIB gene fusions occur in a subset of tumors, 4) high MYB expression correlates with worse patient survival.
PMCID: PMC3225955  PMID: 21976542
Gene fusion; Gene fusion; chromosomal translocations; salivary gland carcinomas; molecular alterations
12.  Primary Colonic -Type Adenocarcinoma of the Base of the Tongue: a Previously Unreported Phenotype 
Human Pathology  2009;40(12):1798-1802.
Primary lingual adenocarcinomas are rare and typically of salivary or seromucinous glands origin. Similarly, metastatic adenocarcinoma from distant primary sites to the tongue is an uncommon event, with only three cases from a colonic primary site reported. We present, for the first time, two primary colonic-type adenocarcinomas of the base of the tongue and discuss their putative origin and the clinicopathologic characteristics.
PMCID: PMC3296116  PMID: 19695679
Base of tongue; Primary adenocarcinoma; Intestinal-type; Metastatic carcinoma
13.  Cell-type dependent biomarkers expression in adenoid cystic carcinoma (ACC): biological and therapeutic implications 
Cancer  2010;116(24):5749-5756.
Adenoid cystic carcinoma (ACC), a rare and progressive salivary malignancy, is characterized by cellular, morphologic and clinical heterogeneity. We hypothesize that its dual cellular composition plays an important role in biomarker evaluation, tumor biological behavior and therapy response.
To investigate the differential localization and expression of c-kit and EGFR proteins, we performed immunohistochemical analysis on tissue arrays of 199 tumors and correlated the results with the clinico-pathological factors. The results show c-kit expression to be limited to the inner ductal epithelial cells and the EGFR expression mainly to the outer myoepithelial cells in the majority of tubular and cribriform patterns. In solid ACC, c-kit was uniformly positive while EGFR was consistently negative. Significant statistical correlation was found between c-Kit expression and a poor 3- year outcome, and EGFR expression with a better 3-year outcome. Our findings underscore the importance of cellular subtypes’ localization of biomarkers in the clinical and therapeutic stratification of patients with this entity.
PMCID: PMC2998592  PMID: 20824717
adenoid cystic carcinoma; cellular localization; c-kit; EGFR
14.  Mutational landscape of aggressive cutaneous squamous cell carcinoma 
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.
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.
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.
PMCID: PMC4367811  PMID: 25303977
Head and neck/oral cancers; Melanoma/skin cancers; Mutagenesis; Oncogenes; Suppressor genes; Somatic alterations and genetic and environmental risk factors; cutaneous squamous cell carcinoma; cSCC; genomics
15.  Rheumatoid arthritis mimicking metastatic squamous cell carcinoma 
Head & Neck Oncology  2011;3:26.
We report a case of a cervical rheumatoid nodule in close relation to the hyoid bone mimicking a metastatic carcinoma. A 74-year-old female with a 15-year history of rheumatoid arthritis (RA) on treatment with methotrexate presented with tenderness of the right base of tongue. Imaging demonstrated a 1.4 cm cystic lesion at the hyoid bone. Biopsies were unsuccessful and the patient required surgical resection of the mass. A trans-cervical approach was used. Pathology revealed a necrotizing granuloma compatible with rheumatoid etiology. The clinician should be aware that, in a patient with a neck mass, in the presence of active RA, rheumatoid nodules should be part of the differential diagnosis.
PMCID: PMC3118955  PMID: 21569598
Rheumatoid; arthritis; nodule; hyoid; larynx
16.  Integrated Genomic Characterization of Papillary Thyroid Carcinoma 
Agrawal, Nishant | Akbani, Rehan | Aksoy, B. Arman | Ally, Adrian | Arachchi, Harindra | Asa, Sylvia L. | Auman, J. Todd | Balasundaram, Miruna | Balu, Saianand | Baylin, Stephen B. | Behera, Madhusmita | Bernard, Brady | Beroukhim, Rameen | Bishop, Justin A. | Black, Aaron D. | Bodenheimer, Tom | Boice, Lori | Bootwalla, Moiz S. | Bowen, Jay | Bowlby, Reanne | Bristow, Christopher A. | Brookens, Robin | Brooks, Denise | Bryant, Robert | Buda, Elizabeth | Butterfield, Yaron S.N. | Carling, Tobias | Carlsen, Rebecca | Carter, Scott L. | Carty, Sally E. | Chan, Timothy A. | Chen, Amy Y. | Cherniack, Andrew D. | Cheung, Dorothy | Chin, Lynda | Cho, Juok | Chu, Andy | Chuah, Eric | Cibulskis, Kristian | Ciriello, Giovanni | Clarke, Amanda | Clayman, Gary L. | Cope, Leslie | Copland, John | Covington, Kyle | Danilova, Ludmila | Davidsen, Tanja | Demchok, John A. | DiCara, Daniel | Dhalla, Noreen | Dhir, Rajiv | Dookran, Sheliann S. | Dresdner, Gideon | Eldridge, Jonathan | Eley, Greg | El-Naggar, Adel K. | Eng, Stephanie | Fagin, James A. | Fennell, Timothy | Ferris, Robert L. | Fisher, Sheila | Frazer, Scott | Frick, Jessica | Gabriel, Stacey B. | Ganly, Ian | Gao, Jianjiong | Garraway, Levi A. | Gastier-Foster, Julie M. | Getz, Gad | Gehlenborg, Nils | Ghossein, Ronald | Gibbs, Richard A. | Giordano, Thomas J. | Gomez-Hernandez, Karen | Grimsby, Jonna | Gross, Benjamin | Guin, Ranabir | Hadjipanayis, Angela | Harper, Hollie A. | Hayes, D. Neil | Heiman, David I. | Herman, James G. | Hoadley, Katherine A. | Hofree, Matan | Holt, Robert A. | Hoyle, Alan P. | Huang, Franklin W. | Huang, Mei | Hutter, Carolyn M. | Ideker, Trey | Iype, Lisa | Jacobsen, Anders | Jefferys, Stuart R. | Jones, Corbin D. | Jones, Steven J.M. | Kasaian, Katayoon | Kebebew, Electron | Khuri, Fadlo R. | Kim, Jaegil | Kramer, Roger | Kreisberg, Richard | Kucherlapati, Raju | Kwiatkowski, David J. | Ladanyi, Marc | Lai, Phillip H. | Laird, Peter W. | Lander, Eric | Lawrence, Michael S. | Lee, Darlene | Lee, Eunjung | Lee, Semin | Lee, William | Leraas, Kristen M. | Lichtenberg, Tara M. | Lichtenstein, Lee | Lin, Pei | Ling, Shiyun | Liu, Jinze | Liu, Wenbin | Liu, Yingchun | LiVolsi, Virginia A. | Lu, Yiling | Ma, Yussanne | Mahadeshwar, Harshad S. | Marra, Marco A. | Mayo, Michael | McFadden, David G. | Meng, Shaowu | Meyerson, Matthew | Mieczkowski, Piotr A. | Miller, Michael | Mills, Gordon | Moore, Richard A. | Mose, Lisle E. | Mungall, Andrew J. | Murray, Bradley A. | Nikiforov, Yuri E. | Noble, Michael S. | Ojesina, Akinyemi I. | Owonikoko, Taofeek K. | Ozenberger, Bradley A. | Pantazi, Angeliki | Parfenov, Michael | Park, Peter J. | Parker, Joel S. | Paull, Evan O. | Pedamallu, Chandra Sekhar | Perou, Charles M. | Prins, Jan F. | Protopopov, Alexei | Ramalingam, Suresh S. | Ramirez, Nilsa C. | Ramirez, Ricardo | Raphael, Benjamin J. | Rathmell, W. Kimryn | Ren, Xiaojia | Reynolds, Sheila M. | Rheinbay, Esther | Ringel, Matthew D. | Rivera, Michael | Roach, Jeffrey | Robertson, A. Gordon | Rosenberg, Mara W. | Rosenthall, Matthew | Sadeghi, Sara | Saksena, Gordon | Sander, Chris | Santoso, Netty | Schein, Jacqueline E. | Schultz, Nikolaus | Schumacher, Steven E. | Seethala, Raja R. | Seidman, Jonathan | Senbabaoglu, Yasin | Seth, Sahil | Sharpe, Samantha | Mills Shaw, Kenna R. | Shen, John P. | Shen, Ronglai | Sherman, Steven | Sheth, Margi | Shi, Yan | Shmulevich, Ilya | Sica, Gabriel L. | Simons, Janae V. | Sipahimalani, Payal | Smallridge, Robert C. | Sofia, Heidi J. | Soloway, Matthew G. | Song, Xingzhi | Sougnez, Carrie | Stewart, Chip | Stojanov, Petar | Stuart, Joshua M. | Tabak, Barbara | Tam, Angela | Tan, Donghui | Tang, Jiabin | Tarnuzzer, Roy | Taylor, Barry S. | Thiessen, Nina | Thorne, Leigh | Thorsson, Vésteinn | Tuttle, R. Michael | Umbricht, Christopher B. | Van Den Berg, David J. | Vandin, Fabio | Veluvolu, Umadevi | Verhaak, Roel G.W. | Vinco, Michelle | Voet, Doug | Walter, Vonn | Wang, Zhining | Waring, Scot | Weinberger, Paul M. | Weinstein, John N. | Weisenberger, Daniel J. | Wheeler, David | Wilkerson, Matthew D. | Wilson, Jocelyn | Williams, Michelle | Winer, Daniel A. | Wise, Lisa | Wu, Junyuan | Xi, Liu | Xu, Andrew W. | Yang, Liming | Yang, Lixing | Zack, Travis I. | Zeiger, Martha A. | Zeng, Dong | Zenklusen, Jean Claude | Zhao, Ni | Zhang, Hailei | Zhang, Jianhua | Zhang, Jiashan (Julia) | Zhang, Wei | Zmuda, Erik | Zou., Lihua
Cell  2014;159(3):676-690.
Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Here, we describe the genomic landscape of 496 PTCs. We observed a low frequency of somatic alterations (relative to other carcinomas) and extended the set of known PTC driver alterations to include EIF1AX, PPM1D and CHEK2 and diverse gene fusions. These discoveries reduced the fraction of PTC cases with unknown oncogenic driver from 25% to 3.5%. Combined analyses of genomic variants, gene expression, and methylation demonstrated that different driver groups lead to different pathologies with distinct signaling and differentiation characteristics. Similarly, we identified distinct molecular subgroups of BRAF-mutant tumors and multidimensional analyses highlighted a potential involvement of oncomiRs in less-differentiated subgroups. Our results propose a reclassification of thyroid cancers into molecular subtypes that better reflect their underlying signaling and differentiation properties, which has the potential to improve their pathological classification and better inform the management of the disease.
PMCID: PMC4243044  PMID: 25417114
17.  Overexpression of the p53 inhibitor MDM4 in Head and Neck Squamous Carcinomas 
Human pathology  2007;38(10):1553-1562.
The p53 tumor suppressor is mutated in the majority of human tumors. MDM2, a well-known inhibitor of p53, is overexpressed in a large number of tumors suggesting that increased levels of MDM2 also contribute to tumorigenesis. A novel p53 inhibitor, MDM4, was more recently identified. The role of MDM4 in cancer development is not well understood. We set out to examine the levels of MDM4 by immunohistochemistry in Head and Neck Squamous Carcinomas (HNSC) to ask whether high MDM4 levels could contribute to its development and progression. In addition, MDM2 and p53 levels were examined to identify overlapping expression patterns. MDM4 is present at high levels in 50% of HNSC. Additionally, overexpression of MDM2 was detected in 80% of tumors, many of which were also positive for MDM4. A subset of tumors displayed high levels of all three proteins. Sequencing of the p53 gene revealed that tumors with positive immunoreactivity for MDM2 or MDM4, some of which also had high levels of p53, did not carry mutations in this gene. Thus, the detection of p53 by immunohistochemistry was not synonymous with the presence of p53 mutations. Expression of both MDM2 and MDM4 in tumors without p53 mutations strongly suggests that MDM2 and MDM4 inhibit the activity of this tumor suppressor in HNSC.
PMCID: PMC2699677  PMID: 17651783
MDM2; p53 mutations; immunohistochemistry
18.  An Attenuated Adenovirus, ONYX-015, As Mouthwash Therapy for Premalignant Oral Dysplasia 
Dysplastic lesions of the oral epithelium are known precursors of oral cancer. A significant proportion of oral dysplastic lesions have functional defects in p53 response pathways. The ONYX-015 adenovirus is selectively cytotoxic to cells carrying defects in p53-dependent signaling pathways. The current study sought to establish the feasibility and activity of ONYX-015 administered topically as a mouthwash to patients with clinically apparent and histologically dysplastic lesions of the oral mucosa.
Patients and Methods
A total of 22 patients (19 assessable patients) were enrolled onto the study. ONYX-015 was administered on three different schedules to consecutive cohorts. Biopsies of the involved mucosa were performed to evaluate histologic response and changes in expression of putative markers of malignant potential, including p53, cyclin D1, and Ki-67. Serology was performed to measure antiadenoviral titers.
Histologic resolution of dysplasia was seen in seven (37%) of 19 patients, and the grade of dysplasia improved in one additional patient. The majority of responses were transient. No toxicity greater than grade 2 (febrile episode in one patient) was observed. Only one of seven patients demonstrated an increase in circulating antiadenoviral antibody titer while on therapy. Although responding and resistant lesions had similar mean p53 staining at baseline, histologic response correlated with a decrease in p53 positivity over time. Significant changes in cyclin D1 or Ki-67 were not observed. Viral replication was confirmed in two of three lesions examined.
This novel approach to cancer prevention is tolerable, feasible, and has demonstrable activity.
PMCID: PMC4523388  PMID: 14597742
19.  Squamous cell carcinoma of the oral tongue in young non-smokers is genomically similar to tumors in older smokers 
Epidemiological studies have identified an increasing incidence of squamous cell carcinoma of the oral tongue (SCCOT) in younger patients.
Experimental Design
DNA isolated from tongue tumors of young (<45 yrs, non-smokers) and old (>45 yrs) patients at was subjected to whole-exome sequencing and copy number analysis. These data were compared to data from similar patients in the The Cancer Genome Atlas (TCGA) project.
In this study, we found that gene-specific mutation and copy number alteration frequencies were similar between young and old SCCOT patients in two independent cohorts. Likewise, the types of base changes observed in the young cohort were similar to those in the old cohort even though they differed in smoking history. TCGA data also demonstrate that the genomic effects of smoking are tumor-site specific, and we find that smoking has only a minor impact on the types of mutations observed in SCCOT.
Overall, tumors from young SCCOT patients appear genomically similar to those of older SCCOT patients, and the cause for the increasing incidence of young SCCOT remains unknown. These data indicate that the functional impact of smoking on carcinogenesis in SCCOT is still poorly understood.
PMCID: PMC4102633  PMID: 24874835
Young tongue cancer; SCCOT; Head and neck/oral cancers; Tobacco
20.  Genetic Variants in DNA Double-Strand Break Repair Genes and Risk of Salivary Gland Carcinoma: A Case-Control Study 
PLoS ONE  2015;10(6):e0128753.
DNA double strand break (DSB) repair is the primary defense mechanism against ionizing radiation-induced DNA damage. Ionizing radiation is the only established risk factor for salivary gland carcinoma (SGC). We hypothesized that genetic variants in DSB repair genes contribute to individual variation in susceptibility to SGC. To test this hypothesis, we conducted a case-control study in which we analyzed 415 single nucleotide polymorphisms (SNPs) in 45 DSB repair genes in 352 SGC cases and 598 controls. Multivariate logistic regression analysis was performed to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Rs3748522 in RAD52 and rs13180356 in XRCC4 were significantly associated with SGC after Bonferroni adjustment; ORs (95% CIs) for the variant alleles of these SNPs were 1.71 (1.40-2.09, P=1.70 × 10-7) and 0.58 (0.45-0.74, P=2.00 × 10-5) respectively. The genetic effects were modulated by histological subtype. The association of RAD52-rs3748522 with SGC was strongest for mucoepidermoid carcinoma (OR=2.21, 95% CI: 1.55-3.15, P=1.25 × 10-5, n=74), and the association of XRCC4-rs13180356 with SGC was strongest for adenoid cystic carcinoma (OR=0.60, 95% CI: 0.42-0.87, P=6.91 × 10-3, n=123). Gene-level association analysis revealed one gene, PRKDC, with a marginally significant association with SGC risk in non-Hispanic whites. To our knowledge, this study is the first to comprehensively evaluate the genetic effect of DSB repair genes on SGC risk. Our results indicate that genetic variants in the DSB repair pathways contribute to inter-individual differences in susceptibility to SGC and show that the impact of genetic variants differs by histological subtype. Independent studies are warranted to confirm these findings.
PMCID: PMC4452711  PMID: 26035306
21.  Gain-of-function mutant p53 but not p53 deletion promotes head and neck cancer progression in response to oncogenic K-ras 
The Journal of pathology  2011;225(4):479-489.
Mutations in p53 occur in over 50% of the human head and neck squamous cell carcinomas (SCCHN). The majority of these mutations result in the expression of mutant forms of p53, rather than deletions in the p53 gene. Some p53 mutants are associated with poor prognosis in SCCHN patients. However, the molecular mechanisms that determine the poor outcome of cancers carrying p53 mutations are unknown. Here, we generated a mouse model for SCCHN and found that activation of the endogenous p53 gain-of-function mutation p53R172H, but not deletion of p53, cooperates with oncogenic K-ras during SCCHN initiation, accelerates oral tumour growth, and promotes progression to carcinoma. Mechanistically, expression profiling of the tumours that developed in these mice and studies using cell lines derived from these tumours determined that mutant p53 induces the expression of genes involved in mitosis, including cyclin B1 and cyclin A, and accelerates entry in mitosis. Additionally, we discovered that this oncogenic function of mutant p53 was dependent on K-ras because the expression of cyclin B1 and cyclin A decreased, and entry in mitosis was delayed, after suppressing K-ras expression in oral tumour cells that express p53R172H. The presence of double-strand breaks in the tumours suggests that oncogene-dependent DNA damage resulting from K-ras activation promotes the oncogenic function of mutant p53. Accordingly, DNA damage induced by doxorubicin also induced increased expression of cyclin B1 and cyclin A in cells that express p53R172H. These findings represent strong in vivo evidence for an oncogenic function of endogenous p53 gain-of-function mutations in SCCHN and provide a mechanistic explanation for the genetic interaction between oncogenic K-ras and mutant p53.
PMCID: PMC4302750  PMID: 21952947
mutant p53; head and neck cancer; mouse model; ras; gain-of-function mutations
22.  Molecular Profiling of Sinonasal Undifferentiated Carcinoma 
Head & neck  2013;36(1):10.1002/hed.23267.
Sinonasal undifferentiated carcinoma remains a poorly characterized malignancy at both the clinical and molecular level, and consequently the optimal treatment strategy remains undefined.
We utilized a mass spectroscopy-based approach (Sequenom™) to evaluate 95 hallmark single nucleotide variations within 12 oncogenes or tumor suppressor genes (AKT, BRAF, CDK4, Beta-catenin, EGFR, FBXW7, JAK2, c-KIT, KRAS, PDGFR, PI3K, VEGF) in 13 histologically confirmed SNUC cases.
None of the samples demonstrated activating mutations in any of the 95 SNVs.
Select clinically relevant activating genomic mutations were not identified the 13 patient samples. However, polymorphisms were noted within the promoter region of VEGF. These may merit future study as predictive biomarkers for treatment response or overall survival. Additionally, future studies focusing on larger tumor sets and utilizing whole genome or exome sequencing may help define genetic aberrations in SNUC that can be clinically targeted with available or emerging biological agents.
PMCID: PMC3874284  PMID: 23633104
SNUC; Sinonasal Undifferentiated Carcinoma; Paranasal sinus tumors; VEGF; Sequenom
23.  Phase II Randomized, Placebo-controlled Trial of Green Tea Extract in Patients with High-risk Oral Premalignant Lesions 
Epidemiologic and preclinical data support the oral-cancer prevention potential of green tea extract (GTE). We randomly assigned patients with high-risk oral premalignant lesions (OPLs) to receive GTE at 500 mg/m2, 750 mg/m2, or 1000 mg/m2 or placebo TID for 12 weeks, evaluating biomarkers in baseline and 12-week biopsies. The OPL clinical response rate was higher in all GTE arms (n=28; 50%) versus placebo (n=11; 18.2%; p=0.09) but did not reach statistical significance. However, the 2 higher-dose GTE arms (58.8% [750, 1000 mg/m2], 36.4% [500 mg/m2], and 18.2%, [placebo], p=0.03) had higher responses, suggesting a dose-response effect. GTE treatment also improved histology (21.4% versus 9.1%, p=0.65), though not statistically significant. GTE was well-tolerated although higher doses increased insomnia/nervousness but produced no grade-4 toxicity. Higher mean baseline stromal VEGF correlated with a clinical (p=0.04) but not histologic response. Baseline scores of other biomarkers (epithelial VEGF, p53, Ki-67, cyclin D1, and p16 promoter methylation) were not associated with a response or survival. Baseline p16 promoter methylation (n=5) was associated with a shorter cancer-free survival. Stromal VEGF and cyclin D1 expression were downregulated in clinically responsive GTE patients and upregulated in non-responsive patients at 12 weeks (versus at baseline). An extended (median 27.5 months) follow-up showed a median time to oral cancer of 46.4 months. GTE may suppress OPLs, in part through reducing angiogenic stimulus (stromal VEGF). Higher doses of GTE may improve short-term (12 week) OPL outcome. The present results support longer-term clinical testing of GTE for oral cancer prevention.
PMCID: PMC4243312  PMID: 19892663
green tea extract; oral premalignant lesions; chemoprevention
24.  Human Papillomavirus and WHO Type I Nasopharyngeal Carcinoma 
The Laryngoscope  2010;120(10):1990-1997.
Nasopharyngeal carcinoma (NPC) is a rare cancer in the United States. An association between NPC and Epstein-Barr virus (EBV) is well-established for World Health Organization (WHO) types II and III (WHO-II/III) NPC but less well-established for WHO type I (WHO-I) NPC. Given the rise in oropharyngeal tumors positive for high-risk human papillomavirus (HPV) and the unique biology of WHO-I NPC, we examined the relationship between HPV and WHO-I NPC.
Study Design
Retrospective case-comparison study.
A search of a large multidisciplinary cancer center tumor registry identified 183 patients seen from January 1999 to December 2008 with incident NPC and no prior cancer. Available paraffin-embedded tumor specimens (N=30) were analyzed for oncogenic HPV status by in-situ hybridization (ISH) and polymerase chain reaction (PCR) for HPV-16 and HPV-18; EBV status by ISH; and p16 expression by immunohistochemistry. Demographic parameters, including race and smoking, were obtained from the medical records.
Among the 18 WHO-I NPC patients, 66% (N=12) were smokers and 17% (N=3) Asian; among the 165 WHO-II/III NPC patients, 44% (N=73) were smokers and 24% (N=39) Asian. Eight WHO-I NPC patients had available paraffin blocks; 5 of 6 were HPV-16-positive by PCR and 4 of 8 were HPV-positive by ISH; only 2 of 8 (25%) were EBV-positive. Twenty-two WHO-II/III NPC patients had available paraffin blocks; only 1 was HPV-positive by ISH, and 13 of 22 (60%) were EBV-positive.
These results suggest that WHO-I NPC is associated with oncogenic HPV, though larger studies are needed to verify these findings.
PMCID: PMC4212520  PMID: 20824783
Human papillomavirus; Nasopharyngeal Carcinoma; WHO Type; In-situ hybridization; Polymerase chain reaction
25.  Origins and functional consequences of somatic mitochondrial DNA mutations in human cancer 
eLife  null;3:e02935.
Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of cancers. Although mitochondria control energy metabolism and apoptosis, the origins and impact of cancer-associated mutations in mtDNA are unclear. In this study, we analyzed somatic alterations in mtDNA from 1675 tumors. We identified 1907 somatic substitutions, which exhibited dramatic replicative strand bias, predominantly C > T and A > G on the mitochondrial heavy strand. This strand-asymmetric signature differs from those found in nuclear cancer genomes but matches the inferred germline process shaping primate mtDNA sequence content. A number of mtDNA mutations showed considerable heterogeneity across tumor types. Missense mutations were selectively neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation undergo negative selection and were almost exclusively heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria and is fundamentally linked to mtDNA replication.
eLife digest
The DNA in a cell's nucleus must be copied faithfully, and divided equally, when a cell divides to produce two new cells. Mistakes—or mutations—are sometimes made during the copying process, and mutations can also be introduced by exposing DNA to damaging agents known as mutagens, such as UV light or cigarette smoke. These mutations are then maintained in all of the descendants of the cell. Most of these mutations have no impact on the cell's characteristics (‘passenger mutations’). However, ‘driver mutations’ that allow cells to divide uncontrollably and spread to other body sites can lead to cancer.
Mitochondria are cellular compartments that are responsible for generating the energy a cell needs to survive and are also responsible for initiating programmed cell death. Mitochondria contain their own DNA—entirely separate from that in the nucleus of the cell—that encodes the proteins most essential for energy production. Mitochondrial DNA molecules are frequently exposed to damaging molecules called reactive oxygen species that are produced by the mitochondria. Therefore, these reactive oxygen species have been thought to be one of the most important causes of mitochondrial DNA mutations. In addition, because cancer cells produce energy differently to normal cells, mutations in the mitochondrial DNA that change the ability of the mitochondria to produce energy have been conventionally thought to help normal cells to become cancerous. However, conclusive evidence for a link between cancer and mitochondrial DNA mutations is lacking.
Ju et al. examined the mitochondrial DNA sequences taken from 1675 cancer biopsies from over thirty different types of cancer and compared these to normal tissue from the same patients. This revealed 1907 mutations in the mitochondrial DNA taken from the cancer cells. The pattern of the mutations suggests that the majority of the mutations are not introduced from reactive oxygen species, but from the errors the mitochondria themselves make in the process of duplicating their DNA when a cell divides. Unexpectedly, known mutagens, such as cigarette smoke or UV light, had a negligible effect on mitochondrial DNA mutations.
Contrary to conventional wisdom, Ju et al. found no evidence that the mitochondrial DNA mutations help cancer to develop or spread. Instead, like passenger mutations found in the DNA in the cell nucleus, most mitochondrial genome mutations have no discernible effect. However, Ju et al. revealed that DNA mutations that damage normal mitochondrial activity are less likely to be maintained in cancer cells. Presumably, mitochondria containing these proteins produce less energy, and so a cell containing too many of these mutations will find it harder to survive. This shows that having enough correctly functioning mitochondria is essential for even cancer cells to thrive.
PMCID: PMC4371858  PMID: 25271376
mitochondrial DNA; somatic mutation; mutational signature; cancer genome; evolution; sequencing; human

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