There are currently few therapeutic options for patients with pancreatic cancer, and new insights into the pathogenesis of this lethal disease are urgently needed. Toward this end, we performed a comprehensive genetic analysis of 24 pancreatic cancers. We first determined the sequences of 23,219 transcripts, representing 20,661 protein-coding genes, in these samples. Then, we searched for homozygous deletions and amplifications in the tumor DNA by using microarrays containing probes for ~10 6 single-nucleotide polymorphisms. We found that pancreatic cancers contain an average of 63 genetic alterations, the majority of which are point mutations. These alterations defined a core set of 12 cellular signaling pathways and processes that were each genetically altered in 67 to 100% of the tumors. Analysis of these tumors' transcriptomes with next-generation sequencing-bysynthesis technologies provided independent evidence for the importance of these pathways and †To whom correspondence should be addressed.

Human cancer is caused by the accumulation of mutations in oncogenes and tumor suppressor genes. To catalog the genetic changes that occur during tumorigenesis, we isolated DNA from 11 breast and 11 colorectal tumors and determined the sequences of the genes in the Reference Sequence database in these samples. Based on analysis of exons representing 20,857 transcripts from 18,191 genes, we conclude that the genomic landscapes of breast and colorectal cancers are composed of a handful of commonly mutated gene "mountains" and a much larger number of gene "hills" that are mutated at low frequency. We describe statistical and bioinformatic tools that may help identify mutations with a role in tumorigenesis. These results have implications for understanding the nature and heterogeneity of human cancers and for using personal genomics for tumor diagnosis and therapy.

Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high density microarrays and sequenced all known protein-coding genes and miRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, 5 to 10-fold fewer than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.

There is considerable debate about the value of personal genome sequencing (1). In addition to the five individuals whose genomes have been sequenced in their entirety, 68 patients have been evaluated for tumor-specific mutations in all exons of protein coding genes (exomic sequencing). This coincidentally yielded information about germline sequence variations in these individuals (2-4). To explore the utility of such information, we evaluated a pancreatic cancer patient (Pa10) whose tumor DNA had been sequenced in (4). This patient had familial pancreatic cancer, as defined by the fact that his sister also had developed the disease.Among the 20, 661 coding genes analyzed, we identified 15,461 germline variants in Pa10 not found in the reference human genome. Of these, 7318 were synonymous, 7721 were missense, 64 were nonsense, 108 were at splice sites, and 250 were small deletions or insertions (54% in-frame). Past studies have shown that tumors arising in patients with a hereditary predisposition harbor no normal alleles of the responsible gene: one allele is inherited in mutant form, often producing a stop codon, and the other (wild type) allele is inactivated by somatic mutation during tumorigenesis. In Pa10, only three genes met these criteria: SERPINB12, RAGE and PALB2. Of these, we considered PALB2 to be the best candidate because germline stop codons in SERPINB12 and RAGE, but not in PALB2, are relatively common in healthy individuals and because germline PALB2 mutations have previously been associated with breast cancer predisposition and Fanconi anemia(5) although its function is not well understood. Pa10 harbored a germline deletion of 4 bp (TTGT at c.172-175) producing a frameshift at codon 58; the pancreatic cancer that developed in Pa10 had somatically acquired a transition mutation (C to T) at a canonical splice site for exon 10 (IVS10+2).To determine whether PALB2 mutations occur in other patients with familial pancreatic cancer, we sequenced this gene in a cohort of 96 familial pancreatic cancer patients, 90 of which were of Caucasian ancestry. Sixteen of these patients had one first degree relative with pancreatic cancer and 80 had at least two additional relatives, at least one of which was first degree, with the disease. Truncating mutations were identified in three of the 96 patients, each producing a different stop codon (Fig. 1). The average age-of-onset of pancreatic cancer in these families was 66.7 years, similar to the mean age of onset of 65.3 years in the families without PALB2 mutations. We determined the germ-line sequence of an affected brother in one of these kindreds, and he harbored the same stop codon. Truncating mutations in PALB2 are rare in individuals without cancer; none have been reported among 1,084 normal individuals in a previous study using a cohort of similar ethnicity to ours (6). While some families we identified with a PALB2 stop mutation had a history of both breast and pancreatic cancer, breast cancer

The incidence of melanoma is increasing more than any other cancer, and knowledge of its genetic alterations is limited. To systematically analyze such alterations, we performed whole-exome sequencing of 14 matched normal and metastatic tumor DNAs. Using stringent criteria, we identified 68 genes that appeared to be somatically mutated at elevated frequency, many of which are not known to be genetically altered in tumors. Most importantly, we discovered that TRRAP harbored a recurrent mutation that clustered in one position (p. Ser722Phe) in 6 out of 67 affected individuals (~4%), as well as a previously unidentified gene, GRIN2A, which was mutated in 33% of melanoma samples. The nature, pattern and functional evaluation of the TRRAP recurrent mutation suggest that TRRAP functions as an oncogene. Our study provides, to our knowledge, the most comprehensive map of genetic alterations in melanoma to date and suggests that the glutamate signaling pathway is involved in this disease.

SUMMARY Protein-DNA interactions (PDIs) mediate a broad range of functions essential for cellular differentiation, function, and survival. However, it is still a daunting task to comprehensively identify and profile sequence-specific PDIs in complex genomes. Here, we have used a combined bioinformatics and protein microarray-based strategy to systematically characterize the human protein-DNA interactome. We identified 17,718 PDIs between 460 DNA motifs predicted to regulate transcription and 4,191 human proteins of various functional classes. Among them, we recovered many known PDIs for transcription factors (TFs). We also identified a large number of new PDIs for known TFs, as well as for previously uncharacterized TFs. Remarkably, we found that over three hundred proteins not previously annotated as TFs also showed sequence-specific PDIs, including RNA binding proteins, mitochondrial proteins, and protein kinases. One of such unconventional DNA-binding proteins, MAPK1, acts as a transcriptional repressor for interferon gamma-induced genes.

Tyrosine phosphorylation is important in signaling pathways underlying tumorigenesis. A mutational analysis of the Protein Tyrosine Kinase (PTK) gene family in cutaneous metastatic melanoma identified 30 somatic mutations in the kinase domain of 19 PTKs. The whole of the coding region of these 19 PTKs was further evaluated for somatic mutations in a total of 79 melanoma samples. This analysis revealed novel ERBB4 mutations in 19% of melanoma patients and that an additional two kinases (FLT1 and PTK2B) are mutated in 10% of melanomas. Seven missense mutations in the most commonly altered PTK (ERBB4) were examined and found to increase kinase activity and transformation ability. Melanoma cells expressing mutant ERBB4 had reduced cell growth after shRNA-mediated knockdown of ERBB4 or treatment with the ERBB inhibitor lapatinib. These studies might lead to personalized therapeutics specifically targeting the kinases that are mutationally altered in individual melanomas.Malignant melanoma is the most fatal skin cancer 1,2 . To develop personalized treatments for advanced disease, it is important to identify genetic alterations leading to melanoma. Protein tyrosine kinases (PTKs) are frequently mutated in cancer (http://www.sanger.ac.uk/genetics/CGP/Census/), and since they are amenable to pharmacologic inhibition 3,4 , further analysis of the PTK gene family may identify new therapeutic strategies. In this study, we used high-throughput gene sequencing to analyze the entire PTK gene family in melanoma, and have identified many novel somatic alterations.We initially sequenced the coding exons comprising the kinase domains of all 86 members of this gene superfamily in 29 melanomas (Supplementary Table 1). A total of 593 exons were extracted from genomic databases and amplified by polymerase chain reaction (PCR) * To whom correspondence should be addressed: National Human Genome Research Institute, 50 South Drive, MSC 8000, Building 50, Room 5140, Bethesda MD 20892-8000, Phone: 301-451-2628, Fax: 301-480-9864, samuelsy@mail.nih.gov. Author contributions T.DP. and Y.S. designed the study; J.R.W. and S.A.R. collected and analyzed the melanoma samples, N.S.A., J.C.C., K.E.Y., J.C.L., NISC., P.C and Y.S. analyzed the genetic data; T.D.P., X.W. and K.E.Y., performed and analyzed the functional data. All authors contributed to the final version of the paper. NIH Public Access Author ManuscriptNat Genet. Author manuscript; available in PMC 2010 July 6. Table 2) and directly sequenced with dye-terminator chemistry. We determined whether a mutation was somatic (i.e., tumor-specific) by examining the sequence of the gene in genomic DNA from normal tissue of the relevant patient. From the ~12 Mb of sequence information obtained, we identified 19 genes containing a total of 30 somatic mutations within their kinase domains. All coding exons of these 19 genes were then analyzed for mutations in a total of 79 melanoma samples using specific primers (Supplementary Table 3).We identified 99 non-synonymous, somatic mutations in ...

Purpose: Recently, the majority of protein coding genes were sequenced in a collection of pancreatic cancers, providing an unprecedented opportunity to identify genetic markers of prognosis for patients with adenocarcinoma of the pancreas. Experimental Design: We previously sequenced more than 750 million base pairs of DNA from 23,219 transcripts in a series of 24 adenocarcinomas of the pancreas. In addition, 39 genes that were mutated in more than one of these 24 cancers were sequenced in a separate panel of 90 well-characterized adenocarcinomas of the pancreas. Of these 114 patients, 89 underwent pancreaticoduodenectomy, and the somatic mutations in these cancers were correlated with patient outcome. Results: When adjusted for age, lymph node status, margin status, and tumor size, SMAD4 gene inactivation was significantly associated with shorter overall survival (hazard ratio, 1.92; 95% confidence interval, 1.20-3.05; P = 0.006). Patients with SMAD4 gene inactivation survived a median of 11.5 months, compared with 14.2 months for patients without SMAD4 inactivation. By contrast, mutations in CDKN2A or TP53 or the presence of multiple (z4) mutations or homozygous deletions among the 39 most frequently mutated genes were not associated with survival. Conclusions: SMAD4 gene inactivation is associated with poorer prognosis in patients with surgically resected adenocarcinoma of the pancreas.Pancreatic cancer is the fourth leading cause of cancer death in the United States (1). Worldwide pancreatic cancer is responsible for more than 213,000 deaths each year (2). The 5-year overall survival rate for all patients diagnosed with pancreatic cancer is less than 4% (3). Surgical resection offers the best hope for long-term survival, with a 17% 5-year survival rate in most surgical series (4 -10). A number of pathologic features have been shown to correlate with outcome following surgery (4,(11)(12)(13)(14). For example, the completeness of resection (margin status), size of the cancer, degree of differentiation, vascular invasion, lymph node status, and tumor stage are all independent prognostic indicators following pancreaticoduodenectomy for pancreatic cancer (4, 11 -16). These factors have been useful guides in the clinical management of patients with pancreatic cancer. Genetic markers that could be used as prognostic indicators of outcome would be useful in establishing an individualized treatment plan for a patient. For example, a more aggressive surgical approach, such as vascular resection and reconstruction, may be considered for a patient with a reduced risk of systemic recurrence.In an attempt to establish such genetic makers, we took advantage of the recently completed mutational analysis of the pancreatic cancer coding genome (17). The results of this ''pancreatic cancer genome project'' provide a unique opportunity to determine if any genes with somatic changes correlate with patient outcome following surgical resection (17). This previous study included the sequencing of the protein-coding exons f...