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Background: A major challenge in interpreting high-throughput multianalyte genomic data sets such as those produced by the ISPY clinical trials is data integration and interpretation within the context of biologically relevant pathways. To address this need, the data analysis tool PARADIGM (PAthway Recognition Algorithm using Data Integration on Genomic Models) was developed to infer the activities of genetic pathways by integrating any number of functional genomic data sets for a given patient sample into a pathway activity profile. Methods: We used PARADIGM to integrate gene expression (Agilent 44K) and DNA copy number data (AFFY 22K and 330K MIP) from 133 ISPY-1 patients into pathway component activity levels for approximately 1400 curated signal transduction, transcriptional and metabolic pathways superimposed onto a single non-redundant ‘SuperPathway'. These pathway activities then become the substrate for statistical analyses to identify pathways characterizing different breast cancer subtypes, as well as those associated with recurrence and response to neoadjuvant chemotherapy within breast cancer subgroups. To identify subtype-specific pathway activities, we used ANOVA for initial feature filtering followed by Tukey analysis with Benjamini Hochberg multiple testing correction. For other binary outcome comparisons we used Mann-Whitney (2-sample Wilcoxon) analysis. PARADIGM results were corroborated with pathway enrichment analysis and filtered for significance. Results: In agreement with breast cancer cell line and other prior studies, basal-like and triple negative cancers are dominated by upregulation of the FOXM1 and MYC/Max subnetworks and downregulation of the FOXA1/ER signal transduction pathway, the converse of the activity pattern seen in luminal breast cancers. These and other subtype associations pass stringent multiple testing corrected significance tests. Though an association study of recurrence over the entire patient cohort mostly yields pathways characteristic of basal-like tumors, alternative pathway associations emerge when subtypes are analyzed individually for outcome and significance tests are relaxed to include features that pass un-corrected Wilcoxon significance tests and also generate highly significant pathway enrichment scores. Subtype-specific drivers of recurrence and chemo-resistance supported by this level of evidence include ALK1/2 (TGFB-BMP) and p53 effector signaling for basals and Syndecan-1 and c-MYC for luminals. Chemo-sensitivity pathways, assessed by association with pCR and RCB1, appear to be subtype-specific as well, with HDAC class 1 signaling, LRP6-Wnt, and IRE1alpha chaperones dominating basal-like cancers and c-MYB activity dominating Her2+ cancers, whereas chemo-sensitivity of HR+Her2- cancers though rare appears to be driven by the DNA damage axis (BRCA/BARD1). Conclusion: These and other similar analyses suggest that patients with TN or basal-like disease might benefit from the addition of ALK1 pathway inhibitors to treatment, whereas high risk HR+ patients might benefit from Syndecan-1 inhibitors. C-MYC/MAX inhibitors might benefit all high risk patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-06-09.

Recent prostate-specific antigen–based screening trials indicate an urgent need for novel and noninvasive biomarker identification strategies to improve the prediction of prostate cancer behavior. Noncoding microRNAs (miRNA) in the serum and plasma have been shown to have potential as noninvasive markers for physiologic and pathologic conditions. To identify serum miRNAs that diagnose and correlate with the prognosis of prostate cancer, we developed a multiplex quantitative reverse transcription PCR method involving the purification of multiplex PCR products followed by uniplex analysis on a microfluidics chip to evaluate 384 human miRNAs. Using Dgcr8 and Dicer knockout (small RNA-deficient) mouse ES cells as the benchmark, we confirmed the validity of our technique and uncovered a considerable lack of accuracy in previously published methods. Profiling 48 sera from healthy men and untreated prostate cancer patients with differing CAPRA scores, we identified miRNA signatures that allow us to diagnose cancer patients and correlate with a prognosis. These serum signatures include oncogenic and tumor-suppressive miRNAs, suggesting functional roles in prostate cancer progression. Cancer Res; 71(2); 550–60. ©2010 AACR.

(Objective) The rising incidence of oral tongue squamous cell carcinoma (OTSCC) in patients who have never smoked and the paucity of knowledge of its biological behavior prompted us to develop a new cell line originating from a never-smoker. (Materials and methods) Fresh tumor tissue of keratinizing OTSCC was collected from a 44-year-old woman who had never smoked. Serum-free media with a low calcium concentration were used in cell culture, and a multifaceted approach was taken to verify and characterize the cell line, designated UCSF-OT-1109.(Results) UCSF-OT-1109 was authenticated by STR DNA fingerprint analysis, presence of an epithelial marker EpCAM, absence of human papilloma virus (HPV) DNA, and SCC-specific microscopic appearance. Sphere-forming assays and mouse xenografts supported its tumorigenic potential. Spectral karyotype (SKY) analysis revealed numerical and structural chromosomal abnormalities. Whole-exome sequencing (WES) identified 46 non-synonymous and 13 synonymous somatic single-nucleotide polymorphisms (SNPs) and one frameshift deletion in the coding regions. Specifically, mutations of CDKN2A, TP53, SPTBN5, NOTCH2, and FAM136A were found in the databases. Copy number aberration (CNA) analysis revealed that the cell line loses chromosome 3p and 9p, but lacks amplification of 3q and 11q (as does HPV-negative, smoking-unrelated OTSCC). It also exhibits four distinctive focal amplifications in chromosome 19p, containing 131 genes without SNPs. Particularly, 52 genes showed >3- to 4-fold amplification and could be potential oncogenic drivers.(Conclusion) We have successfully established a novel OTSCC cell line from a never-smoking patient. UCSF-OT-1109 is potentially a robust experimental model of OTSCC in never-smokers. Citation Format: Steven J. Wang, Saurabh Asthana, Annemieke van Zante, Adam B. Olshen, Frank McCormick, Osamu Tetsu. Establishment of an oral tongue squamous cell carcinoma cell line from a never-smoking patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3104.

Most gene expression profiling studies of mesothelioma have been based on relatively small sample numbers, limiting their statistical power. We did Affymetrix U133A microarray analysis on 99 pleural mesotheliomas, in which multivariate analysis showed advanced-stage, sarcomatous histology and P16/CDKN2A homozygous deletion to be significant independent adverse prognostic factors. Comparison of the expression profiles of epithelioid versus sarcomatous mesotheliomas identified many genes significantly overexpressed among the former, including previously unrecognized ones, such as uroplakins and kallikrein 11, both confirmed by immunohistochemistry. Examination of the gene expression correlates of survival showed that more aggressive mesotheliomas expressed higher levels of Aurora kinases A and B and functionally related genes involved in mitosis and cell cycle control. Independent confirmation of the negative effect of Aurora kinase B was obtained by immunohistochemistry in a separate patient cohort. A role for Aurora kinases in the aggressive behavior of mesotheliomas is of potential clinical interest because of the recent development of small-molecule inhibitors. We then used our data to develop microarray-based predictors of 1 year survival; these achieved a maximal accuracy of 68% in cross-validation. However, this was inferior to prognostic prediction based on standard clinicopathologic variables and P16/CDNK2A status (accuracy, 73%), and adding the microarray model to the latter did not improve overall accuracy. Finally, we evaluated three recently published microarray-based outcome prediction models, but their accuracies ranged from 63% to 67%, consistently lower than reported. Gene expression profiling of mesotheliomas is an important discovery tool, but its power in clinical prognostication has been overestimated. (Cancer Res 2006; 66(6): 2970-9)

Adult male germ cell tumors (GCTs) comprise distinct groups: seminomas and nonseminomas, which include pluripotent embryonal carcinomas as well as other histologic subtypes exhibiting various stages of differentiation. Almost all GCTs show 12p gain, but the target genes have not been clearly defined. To identify 12p target genes, we examined Affymetrix (Santa Clara, CA) U133A+B microarray (∼83% coverage of 12p genes) expression profiles of 17 seminomas, 84 nonseminoma GCTs, and 5 normal testis samples. Seventy-three genes on 12p were significantly overexpressed, including GLUT3 and REA (overexpressed in all GCTs) and CCND2 and FLJ22028 (overexpressed in all GCTs, except choriocarcinomas). We characterized a 200-kb gene cluster at 12p13.31 that exhibited coordinated overexpression in embryonal carcinomas and seminomas, which included the known stem cell genes NANOG, STELLA, and GDF3 and two previously uncharacterized genes. A search for other coordinately regulated genomic clusters of stem cell genes did not reveal any genomic regions similar to that at 12p13.31. Comparison of embryonal carcinoma with seminomas revealed relative overexpression of several stem cell–associated genes in embryonal carcinoma, including several core “stemness” genes (EBAF, TDGF1, and SOX2) and several downstream targets of WNT, NODAL, and FGF signaling (FGF4, NODAL, and ZFP42). Our results indicate that 12p gain is a functionally relevant change leading to activation of proliferation and reestablishment/maintenance of stem cell function through activation of key stem cell genes. Furthermore, the differential expression of core stem cell genes may explain the differences in pluripotency between embryonal carcinomas and seminomas. (Cancer Res 2006; 66(2): 820-7)

Mutational hotspots indicate selective pressure across a population of tumor samples and drive the dysregulation of proliferation, invasion, and apoptosis in many human tumors. We recently performed a comprehensive analysis of hotspot mutations in 11,000 retrospectively characterized primary untreated cancers, revealing widespread lineage diversity and mutational specificity. Yet, as broad-based clinical sequencing has begun to guide the routine care of patients, most clinically sequenced cancer patients present with recurrent or metastatic solid tumors. Furthermore, such tumors likely possess distinct mutational patterns driven by prior treatment exposure or treatment-associated evolution after diagnosis that are undetectable in primary untreated samples. We therefore sought to expand our analysis to incorporate such prospectively characterized active cancer patients as part of an ongoing initiative at Memorial Sloan Kettering Cancer Center. With nearly 10,000 patients profiled thus far, this growing prospective dataset with extensive clinical and treatment history is a transformative resource and the largest of its kind. Here, we combined these prospective data with retrospective studies to analyze the somatic mutational landscape of 21,000 cancer patients across 74 cancer types representing the broadest range of human malignancies characterized to date. We developed and applied a biologically and statistically principled computational model to identify hotspots mutated more frequently than would be expected in the absence of selection. This analysis uncovered hundreds of new biologically and therapeutically relevant candidate driver mutations for which targeted agents are currently being investigated in early phase trials. Indeed, we identify several hotspots in key effectors of PI3K and MAPK signaling with differential response in vitro to current investigational therapies suggesting mutant allele-specific therapeutic decisions may be warranted. In total, over half of all hotspot mutations identified were novel, including several novel hotspots in small GTPase RAC1 that are lineage specific and functionally distinct. Understanding the distinct molecular function of these mutations, and the specific mutant alleles at individual codons, is a necessary translational prerequisite for enabling precision oncology through clinical decision support for patients sequenced at the point of care. Citation Format: Matthew T. Chang, Sizhi Paul Gao, Tripti Shrestha Bhattarai, Tambudzai Shamu, Cyriac Kandoth, Saurabh Asthana, Jocelyn S. Chapman, JianJiong Gao, Nicholas D. Socci, Adam B. Olshen, David M. Hyman, Michael F. Berger, David B. Solit, Nikolaus D. Schultz, Barry S. Taylor. Identifying novel recurrent mutations reveals candidate actionable mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4366.

Desmoplastic melanomas (DMs) comprise 4% of the overall melanoma burden and have a 5-year survival rate of 85%. DMs are dermal tumors characterized by spindled melanocytes situated within abundant desomplastic stroma. These unusual histological features commonly lead to misdiagnosis. Currently, there are no known genetic drivers. A better understanding of the underlying biology of desmoplastic melanoma would provide biomarkers and therapeutic opportunities. Towards this goal, we performed low-coverage genome and high-coverage exome sequencing of 20 DMs in a discovery cohort, followed by targeted sequencing of 293 candidate genes on a validation cohort of 42 cases. Additionally, high-resolution aCGH was performed on samples from both cohorts. A high mutation burden (median 62 mutations/Mb) ranked desmoplastic melanoma among the most highly mutated cancers sequenced to date. Mutation patterns strongly indicate that UV-radiation is the dominant mutagen and implicate a superficially located cell of origin despite their predominantly intradermal presentation. Novel alterations included recurrent promoter mutations and amplification of NF-kappa B inhibitor epsilon, NFKBIE (IkBϵ) in 14.5% of samples. The promoter mutations typically affect both alleles and occur over a highly conserved DNA region. The mutations are predicted to disrupt a canonical Ets Like Factor 1 (ELF1) binding site. In total, these data imply aberrant NF-kappa B signaling as a pathogenic feature of desmoplastic melanoma. Commonly mutated oncogenes in melanomas, in particular BRAF V600E and NRAS Q61K/R, were absent. Instead, other genetic alterations known to activate the MAPK and PI3K signaling cascades were identified in 73% of samples, affecting NF1, CBL, ERBB2, MAP2K1, MAP3K1, BRAF, EGFR, PTPN11, MET, RAC1, SOS2, NRAS, and PIK3CA. Rb and p53 pathway alterations occurred respectively in 71% and 66% of tumors, affecting RB1, FBXW7, CDK4, PPP6C, CCND1, CDKN2A, TP53, and MDM2. Finally, TERT promoter mutations or amplifications occurred in 90% of tumors. The consequences of the mutations on protein expression levels was confirmed by immunostaining for NF1, EGFR, Rb, CDK4, CCND1, p16, p53, and Mdm2. Collectively, many of these oncogenic mutations are potentially druggable. In conclusion, desmoplastic melanomas harbor distinct genetic alterations that explain their unique biology, and this study illuminates genetic biomarkers and nominates targets for therapeutic intervention. Citation Format: Alan H. Shain, Maria Garrido, Thomas Botton, Eric Talevich, Iweh Yeh, Zack Sanborn, Jongsuk Chung, Nicholas Wang, Hojabr Kakavand, Graham Mann, John Thompson, Thomas Wiesner, Ritu Roy, Adam Olshen, Alexander Gagnon, Joe Gray, Nam Huh, Joe Hur, Klaus Busam, Richard Scolyer, Raymond Cho, Rajmohan Murali, Boris Bastian. Exome sequencing of desmoplastic melanoma reveals recurrent NFKBIE promoter mutations and diverse MAPK/PI3K pathway activating mutations. [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 2968. doi:10.1158/1538-7445.AM2015-2968

The phosphatidylinositol 3'-kinase/Akt pathway is activated frequently in human cancer, and has been implicated in tumor proliferation, cell survival, and resistance to apoptotic stimuli. Akt forms a complex with heat shock protein (Hsp) 90 and Cdc37, and inhibitors of Hsp90 cause Akt degradation. 17-allylamino-17-demethoxygeldanamycin (17-AGG) is an Hsp90 inhibitor currently in Phase I clinical trial. 17-AAG inhibits Akt activation and expression in tumors, and has antitumor activity in breast cancer xenografts. The combination of 17-AAG and Taxol is synergistic, and 17-AAG sensitizes tumor cells to Taxol-induced apoptosis in a schedule-dependent manner. Transfection of membrane-bound p110 PI3k prevented 17-AAG inactivation of Akt and abrogated the enhancement of Taxol-induced apoptosis caused by the drug. 17-AAG and Taxol could be administered together at their maximally tolerated doses to tumor-bearing mice. Doses of 17-AAG that induce HER2 degradation and cause Akt inactivation but have no single agent activity were effective in sensitizing tumors to Taxol. Enhancement was schedule-dependent and maximal when Taxol and 17-AAG were administered on the same day. These results suggest that Hsp90 inhibitors can effectively suppress Akt activity in animal models of human cancer at nontoxic doses, thus sensitizing tumor cells to proapoptotic stimuli.

Genomic abnormalities at 348 loci encoding genes that may contribute to lung cancer transformation and progression were assessed using array comparative genomic hybridization in 21 squamous carcinomas (SqCas) and 16 adenocarcinomas (AdCas). Hierarchical clustering showed a clear pattern of gains and losses for the SqCas, whereas the pattern for AdCas was less distinct. Cross-validated classification using a K-nearest-neighbor assigned, on average, 32 of 37 samples to their proper histological subtype. The most noticeable differences between SqCas and AdCas were gain of chromosome 3q22-q26 and loss of chromosome 3p. These occurred almost exclusively in SqCas. The region of recurrent increase is approximately 30 Mb in extent, ranging from EVI1 to TFRC. PIK3CA, the alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is in this region. The PIK3CA copy number increase was validated using fluorescence in situ hybridization to lung cancer tissue microarrays. Activity of the downstream PI3K effector protein kinase B (PKB) was higher in SqCas than in AdCas and was correlated with PIK3CA copy number (r = 0.75), suggesting that these copy number increases contribute to activation of PI3K signaling in SqCas of the lung.

Array-based comparative genomic hybridization (CGH) uses multiple genomic clones arrayed on a slide to detect relative copy number of tumor DNA sequences. Application of array CGH to tumor specimens makes genetic diagnosis of cancers possible and may help to differentiate relevant subsets of tumors, biologically and clinically, which would allow better prognostic and therapeutic decision making. In this study, we have used array-based CGH to detect DNA copy number alterations in distinct types of renal cell carcinomas for diagnostic purposes. We were able to correctly diagnose 33 of 34 malignant tumors by automated computational means and to group together eight benign neoplasms and normal kidney samples. These results indicate that array-based CGH is capable of diagnosing the vast majority of renal cell carcinomas based on their genetic profiles.

Introduction: The disease course of patients with diffuse low-grade gliomas (LGGs) is notoriously unpredictable. Detailed analysis of the genetic make-up is therefore key to understand this wide variety in overall survival of patients diagnosed with LGGs. Reports on the prognostic value of CNAs other than 1p/19q co-deletion are conflicting, which may be explained by spatial copy number heterogeneity. Aim: Prognostic implications of spatial and temporal copy number aberrations in LGGs by whole genome sequencing. Methods: Approximately 25% of LGG patients have a life expectation of more than 20 years following diagnosis, which necessitates collection of samples with long clinical follow up information. Therefore archival material was selected for this project and WGS was developed to access these samples without the requirement for a normal reference. We collected 156 formalin-fixed and paraffin-embedded (FFPE) samples in a discovery cohort of 98 LGG patients with extensive clinical follow-up data, which included recurrent tumours and spatially distinct regions. An independent cohort of 126 samples was obtained from a recently published study (Alentorn et al, Neuro-oncology 2014). Technical challenges for genome-wide inference of copy number aberrations include repetitive and common DNA sequences in the genome and sequence variation across the general population, as well as the compromised and variable quality of DNA obtained from archived tissues. We developed a robust and cost effective method that infers copy number aberrations from WGS data of approximately 0.1x coverage, without the need for a reference signal. This method implements (1) a combined LOESS correction for mappability and GC content, and improves on previous methods by (2) comprehensive filtering based on public genome project data, 1000-Genomes project and ENCODE blacklists. Results: Both prognostic value, temporal evolution and spatial heterogeneity of CNA were assessed by WGS. We confirmed prognostic favourable value of 1p/19q co-deletion, and demonstrated loss of 10q to be an unfavourable marker. In paired recurrences 10q loss was invariably maintained and surfaced in 4 additional recurrences of the discovery cohort. In spatial regions of LGGs we recognized extensive copy number heterogeneity; 15 of 17 LGGs show spatial variability of CNAs. 1p/19q co-deletion is homogeneous, while loss of 10q is heterogeneously present. Conclusions: we present clinically relevant CNAs, but also demonstrate extensive spatial copy number heterogeneity in diffuse LGGs that might complicate unequivocal biomarker discovery. Citation Format: Ilari Scheinin, Hinke F. van Thuijl, Daoud Sie, Hendrik F. van Essen, Paul P. Eijk, Francois Rustenburg, Ahmed Idbaih, Agusti Alentorn, Gerrit A. Meijer, Mark A. van der Wiel, Henrik Bengtsson, Adam Olshen, Eleonora Aronica, Jan J. Heimans, Jaap C. Reijneveld, Pieter Wesseling, Donna G. Albertson, Dan Pinkel, Bauke Ylstra. A novel approach to copy number assessment by whole genome sequencing reveals extensive spatial heterogeneity in diffuse low-grade glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3426. doi:10.1158/1538-7445.AM2014-3426

Individual bacteria and shifts in the composition of the microbiome have been associated with human diseases including cancer. To investigate changes in the microbiome associated with oral cancers, we profiled cancers and anatomically matched contralateral normal tissue from the same patient by sequencing 16S rDNA hypervariable region amplicons. In cancer samples from both a discovery and a subsequent confirmation cohort, abundance of Firmicutes (especially Streptococcus) and Actinobacteria (especially Rothia) was significantly decreased relative to contralateral clinically normal samples from the same patient. Significant decreases in abundance of these phyla were observed for pre-cancers, but not when comparing samples from contralateral sites (tongue and floor of mouth) from healthy individuals. Patients with cancer or dysplasia, however, could be distinguished from healthy normal individuals by increased abundance of Prevotella. Weighted UniFrac principal coordinates analysis based on 12 taxa separated most cancers from other samples with greatest separation of node positive cases. Citation Format: Donna G. Albertson, Justin Kuczynski, Aditi Bhattacharya, Bing Huey, Patricia M. Corby, Erica L. S. Queiroz, Kira Nightingale, Alexander R. Kerr, Mark D. DeLacure, Ratna Veeramachaneni, Adam Olshen, Brian L. Schmidt. Changes in abundance of oral microbiota associated with oral cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4885. doi:10.1158/1538-7445.AM2014-4885

Deregulated cell proliferation is a defining feature of cancer cells. Normally, an intricate regulatory network ensures proper cell cycle progression during development and for tissue homeostasis. Although many steps in gene regulation imbue specificity to this program, the translational landscape of gene expression underlying cell cycle progression remains poorly understood. To address this outstanding question, we have conducted the first genome-wide analysis of translational regulation using ribosome profiling during each phase of the cell cycle. We have uncovered widespread translational regulation of hundreds of mRNAs that provides a rapid, robust, and precise mechanism for gene regulation that underlies the cell cycle program. These include a number of genes involved in key biological process and hallmarks of cancer cell growth and tumor development including cellular proliferation, cellular metabolism, and safeguarding genomic integrity. We have further characterized a novel network of translational regulation coordinating the expression of the cohesin complex, which is important for proper chromosomal segregation, regulating transcription, and has recently been identified as a common target of mutation in a number of tumor types. Mechanistically, we show that the 5'UTRs of several cohesin mRNAs are sufficient to direct their cell cycle dependent pattern of expression. Thereby, our studies highlight an intricate relationship in translational control between the machineries responsible for maintaining the organization of the genome and protein synthesis. Moreover, we discovered that translational specificity during cell cycle phases coordinates the mTOR signaling pathway. Specifically, we uncovered that translation of RICTOR, a key component of mTORC2, is induced during S-phase, resulting in an increase in mTORC2 signaling during this phase of the cell cycle. Furthermore, we showed that rictor-/- MEFs exhibited a marked decrease in S-phase cells compared to wild-type MEFs, suggesting that RICTOR may be required to prevent premature exit from S-phase, ensuring that cells complete DNA replication. Together, our findings pinpoint that translational regulation of RICTOR underlies cell cycle-dependent mTORC2 activity, which may be a critical determinant of the mammalian cell cycle program. Our findings highlight both the prevalence and dynamic nature of translational regulation during cell cycle progression. This work further suggests a multifaceted mechanism for differential regulation of transcript-specific translational control in the accurate execution of distinct steps of the cell cycle. Current studies are unraveling novel cis-regulatory sequences underlying the molecular mechanisms governing the translational regulation of mRNA networks during the cell cycle and determining how distinct oncogenic insults, which directly impact the translational machinery, alter the expression patterns of these networks leading to uncontrolled cell cycle progression. Citation Format: Craig R. Stumpf, Melissa V. Moreno, Adam B. Olshen, Barry S. Taylor, Davide Ruggero. Plasticity of the translational landscape during the mammalian cell cycle: Implications and new insights for cancer cell proliferation. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A57.

The biologic behavior of oral dysplasia (precancers) and squamous cell carcinoma (SCC) is unpredictable, the main problems in management being identification of patients at risk for metastasis or risk of progression of dysplasias to cancer. Neck metastasis, the most significant clinical factor causing death in oral SCC, reduces survival by 50%. We utilized array comparative genomic hybridization (CGH) to clarify the role of genomic aberrations in oral cancer progression and metastasis. Two oral SCC cohorts: cohort#1, n=89 (Snijders et al, 2005)) and cohort#2, n=63 (with associated pathologic nodal status and 5 year clinical follow up) were profiled using BAC CGH arrays. Two dysplasia cohorts, one with no association to cancer (cohort D1, n=29) and one associated with previous or subsequent cancer (cohort D2, n=10) were also profiled for recurrent copy number aberrations using array CGH. Associations with clinical characteristics were studied and results confirmed in an independent SCC cohort (cohort#3, n=14, Smeets et al., 2009). All samples used in this study were restricted to oral cavity sites only and were archival formalin fixed paraffin embedded tissue. Copy number alterations distinguished two oral dysplasia and cancer subtypes. The presence of one or more of the chromosomal aberrations +3q24-qter, -8pter-p23.1, +8q12-q24.2, and +20 distinguished a major subgroup (70%-80% of lesions, termed 3q8pq20 subtype) from the remainder (20%-30% of lesions, non-3q8pq20). Most notably, 3q8pq20 and non-3q8pq20 subtypes differed significantly in clinical outcome with risk for cervical (neck) lymph node metastasis almost exclusively associated with the 3q8pq20 subtype in two independent oral SCC cohorts (cohort#2 and cohort#3). As metastasis to the cervical lymph nodes is a major determinant of survival in oral SCC patients, our findings indicate that chromosomal aberrations +3p, -8p, +8q and +20 provide a potential biomarker to identify patients at no or low risk of metastasis. The negative predictive value (NPV) i.e. the ability to predict N0 cases at biopsy was 93% for cohort#2 and 100% in cohort#3. Currently, almost all patients diagnosed with oral SCC are treated with comprehensive neck dissection at the time of tumor resection, as salvage of patients who subsequently develop neck metastasis is poor. We propose that the +3q, -8p, +8q and +20 signature is suitable for stratifying patients for risk of metastasis prior to surgery, and so can guide surgical treatment for one of the most challenging treatment decisions - how to treat patients with clinically node-negative (N0) necks. Identification of patients with low risk for metastasis would spare them additional major surgery with its risks and morbidity, as well as reduce medical costs. 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 5079. doi:1538-7445.AM2012-5079

Pancreatic ductal adenocarcinoma (PDAC) is an invariably lethal disease. Due to its late clinical presentation, surgical cure is attempted in only a minority, and resected patients usually recur quickly with incurable, metastatic disease. These attributes may reflect the variable and often disappointing responses seen when deploying the standard treatment, gemicitabine alone or in combination with other chemotherapeutic agents in unselected PDAC populations. Studies in other solid tumors have shown that heterogeneity in therapeutic responses can be anticipated by molecular differences between tumors, and targeting drugs specific to tumor subtypes in which they are predicted to be selectively effective can indeed improve treatment. Seeking to extend this new paradigm of personalized medicine to PDAC, we performed an integrative analysis of transcriptional profiles of human primary PDAC tumors (n = 143) and cell lines (n = 35). We have defined 62 gene (PDAssigner) signature that classifies the human PDAC tumors into three subtypes (classical, QM-PDAC and exocrine-like tumors) and present evidence that two (classical and QM-PDAC) of these three are present in human and mouse PDAC cell lines. Interestingly, we found that PDAC subtype classification is an independent predictor of overall survival, and patients with classical PDAC subtype tumors have significantly better survival than those with QM-PDA tumors. In addition, the expression of GATA6 (a transcriptional factor involved in the embryonic pancreas development) and KRAS (mutated in majority of PDACs) are functionally associated with classical PDAC subtype relative to QM-PDAC subtype. Intriguingly, treatment of PDAC cell lines with gemcitabine or erlotinib shows significantly differential and opposing responses between classical and QM-PDAC subtypes. Overall, we describe here a broadly integrative approach to PDAC subtype discovery and analysis that identifies gene expression signature with prognostic significance in primary patient samples and couples them with biological information and subtype specific drug responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-24. doi:10.1158/1538-7445.AM2011-LB-24

The 5-year survival rate for oral squamous cell carcinoma (SCC) patients at 40% has not improved over the past 40 years. Since genomes of 75-80% of oral SCCs are characterized by regions of gains and losses, we investigated whether copy number aberrations could be used to address two important problems in oral cancer management - identification of (a) tumors with metastatic potential and (b) dysplastic lesions at risk for progression to cancer. Neck metastasis, the most significant clinical factor responsible for death from oral cancer reduces survival by 50%. To determine if copy number aberrations would distinguish tumors with metastatic potential, we carried out array comparative genomic hybridization (CGH) on 26 N0 and 38 N+ cases with at least 5-year follow up (diagnosed between 1997 and 2005). In order to reduce the multiple testing correction, regions of recurrent aberration were identified in an independent set of 89 oral SCCs (Snijders et al. 2005). A comparison of the frequency of aberrations affecting these regions in the N0 and N+ cases revealed no significant differences between the N0 and N+ cases after correction for multiple testing. We used a similar approach to determine if there are significant copy number differences between oral dysplasia and oral SCC. Most oral SCCs are preceded by precancerous lesions, characterized by varying degrees of dysplasia (graded from mild to severe). Transformation to oral SCC is associated with 16% of mild and 55% of moderate/severe dysplasia. Copy number profiles for 44 dysplasia samples comprised of approximately equal numbers of cases graded mild, moderate or severe were obtained by array CGH. Although fewer copy number changes were observed in dysplasia compared to SCC, gains of 3q, 8q and/or chromosome 20 were present at approximately equal frequencies in dysplasia and SCC. In addition, the rare narrow amplicons, characteristic of oral SCC (Snijders et al. 2005), were present in dysplasia. To identify copy number alterations distinguishing dysplasia and SCC, regions of recurrent aberration were defined in an independent set of oral SCCs (the 64 cases from the metastasis study described above). The frequency of these copy number alterations was compared between the dysplasia and the 89 SCC cases (Snijders et al. 2005). Gain of 7p, including EGFR in SCC was found to be significant after correction for multiple testing. This observation is consistent with previous reports of increased expression of EGFR with progression of dysplasia to cancer (Garnis et al., 1998). Since 20-25% of oral SCC harbor almost no copy number changes, suggesting that dysplasia lacking copy number changes may have potential to progress to SCC with chromosomally stable genomes, we suggest that measurement of EGFR expression and/or other genes on 7p may provide a more effective biomarker for dysplasia progression than 7p copy number. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2137.