Mutations In Human Cancers Research Articles

PI3 Kinase, Phospholipase C (PLC)-γ, and RasGRPs Act Cooperatively to Activate the Ras-Extracellular-Related Kinase (ERK) Pathway in Response to Cytokines in Normal and Kras Mutant Myeloid Cells.

Abstract 2512Poster Board II-489Introduction: KRAS is the most common gain-of-function mutation in human cancer and occur in some myeloid malignancies. Despite extensive data implicating aberrant responsiveness to cytokines in the pathogenesis of myeloid leukemias with hyperactive Ras, the underlying mechanisms are largely unknown. We investigated the biochemical basis of hypersensitivity to granulocyte macrophage colony stimulating factor (GM-CSF), IL-3, and stem cell factor (SCF) in primary cells from Mx1-Cre, KrasG12D mice. Methods: We utilized conventional biochemical techniques, phospho-specific flow cytometry, and small molecule inhibitors to interrogate signaling downstream of the activated GM-CSF and c-Kit receptors. Results: We found that endogenous KrasG12D expression deregulates multiple effectors in primary myeloid lineage cells. Although Ras-GTP levels are markedly elevated in bone marrow cells from KrasG12D mice, these cells only fully activate the Raf/MEK/ERK cascade upon cytokine stimulation. We used a “chemical genetic” strategy to explore the biochemical mechanism linking the activated GM-CSF receptor to oncogenic K-RasG12D and ERK. Surprisingly, inhibitors of type I PI3 kinases (PI-90, PI-103) as well as a p110 γ/α isoform inhibitor (AS605240) interfered with GM-CSF and SCF induced Ras/MAPK activation in wild-type and KrasG12D myeloid cells. These effects of PI3 kinase are mediated through activation of PLC-γ and the RasGRP guanine nucleotide exchange factors. Functional studies confirmed that inhibiting PI3 kinase or PLC-γ blocked KrasG12D-mediated myeloid progenitor colony growth. This PI3 kinase/PLC-γ module also functions downstream of the activated c-kit receptor in lineage- cKit+ Sca1+ (LSK) cells in SCF-induced ERK activation. Conclusions: PI3 kinase, PLC-γ, and RasGRPs function upstream of normal and oncogenic K-Ras to efficiently activate the Raf/MEK/ERK cascade in primary hematopoietic cells. These data infer that PI3 kinase is both an important upstream regulator as well as a major downstream effector of Ras-GTP. Significance: Targeting PI3 kinase in hematologic cancers characterized by hyperactive Ras may have unanticipated biochemical and functional consequences in malignant and normal hematopoietic cells. Disclosures:No relevant conflicts of interest to declare.

TP53 Mutations in Human Cancers: Origins, Consequences, and Clinical Use

Somatic mutations in the TP53 gene are one of the most frequent alterations in human cancers, and germline mutations are the underlying cause of Li-Fraumeni syndrome, which predisposes to a wide spectrum of early-onset cancers. Most mutations are single-base substitutions distributed throughout the coding sequence. Their diverse types and positions may inform on the nature of mutagenic mechanisms involved in cancer etiology. TP53 mutations are also potential prognostic and predictive markers, as well as targets for pharmacological intervention. All mutations found in human cancers are compiled in the IARC TP53 Database (http://www-p53.iarc.fr/). A human TP53 knockin mouse model (Hupki mouse) provides an experimental model to study mutagenesis in the context of a human TP53 sequence. Here, we summarize current knowledge on TP53 gene variations observed in human cancers and populations, and current clinical applications derived from this knowledge.

COSMIC (the Catalogue of Somatic Mutations in Cancer): a resource to investigate acquired mutations in human cancer

The catalogue of Somatic Mutations in Cancer (COSMIC) (http://www.sanger.ac.uk/cosmic/) is the largest public resource for information on somatically acquired mutations in human cancer and is available freely without restrictions. Currently (v43, August 2009), COSMIC contains details of 1.5-million experiments performed through 13 423 genes in almost 370 000 tumours, describing over 90 000 individual mutations. Data are gathered from two sources, publications in the scientific literature, (v43 contains 7797 curated articles) and the full output of the genome-wide screens from the Cancer Genome Project (CGP) at the Sanger Institute, UK. Most of the world’s literature on point mutations in human cancer has now been curated into COSMIC and while this is continually updated, a greater emphasis on curating fusion gene mutations is driving the expansion of this information; over 2700 fusion gene mutations are now described. Whole-genome sequencing screens are now identifying large numbers of genomic rearrangements in cancer and COSMIC is now displaying details of these analyses also. Examination of COSMIC’s data is primarily web-driven, focused on providing mutation range and frequency statistics based upon a choice of gene and/or cancer phenotype. Graphical views provide easily interpretable summaries of large quantities of data, and export functions can provide precise details of user-selected data.

A Cancer-associated Aurora A Mutant Is Mislocalized and Misregulated Due to Loss of Interaction with TPX2

Mutations in protein kinases can drive cancer through alterations of the kinase activity or by uncoupling kinase activity from regulation. Changes to protein expression in Aurora A, a mitotic Ser/Thr kinase, are associated with the development of several human cancers, but the effects of somatic cancer-associated mutations have not been determined. In this study we show that Aurora A kinase activity is altered in different ways in three somatic cancer-associated mutations located within the catalytic domain; Aurora A(V174M) shows constitutively increased kinase activity, Aurora A(S155R) activity is decreased primarily due to misregulation, and Aurora A(S361*) activity is ablated due to loss of structural integrity. These alterations suggest vastly different mechanisms for the role of these three mutations in human cancer. We have further characterized the Aurora A(S155R) mutant protein, found that its reduced cellular activity and mislocalization are due to loss of interaction with TPX2, and deciphered the structural basis of the disruption at 2.5 A resolution. Previous studies have shown that disruption of the Aurora A/TPX2 interaction results in defective spindles that generate chromosomal abnormalities. In a panel of 40 samples from microsatellite instability-positive colon cancer patients, we found one example in which the tumor contained only Aurora A(S155R), whereas the normal tissue contained only wild-type Aurora A. We propose that the S155R mutation is an example of a somatic mutation associated with this tumor type, albeit at modest frequency, that could promote aneuploidy through the loss of regulated interactions between Aurora A and its protein partners.

Novel gene rearrangements in transformed breast cells identified by high-resolution breakpoint analysis of chromosomal aberrations

Chromosomal copy number alterations and chromosomal rearrangements are frequent mutations in human cancer. Unlike copy number alterations, little is known about the role and occurrence of chromosomal rearrangements in breast cancer. This may be due to the fact that chromosome-based breakpoint analysis is widely restricted to cultured cells. In order to identify gene rearrangements in breast cancer, we studied the chromosomal breakpoints in radiation-transformed epithelial breast cell lines using a high-resolution array-based approach using 1 Mb bacterial artificial chromosome (BAC) arrays. The breakpoints were further narrowed down by fluorescence in situ hybridisation (FISH) with clones from the 32 k BAC library. The analysis of the cell lines B42-11 and B42-16 revealed rearrangements of chromosomes 7, 8, 10 and 12. We identified the genes Has2, Grid1, Ret, Cpm, Tbx3, Tbx5, Tuba1a, Wnt1 and Arf3 within the breakpoint regions. Quantitative RT-PCR showed a deregulated expression of all of these candidate genes except for Tbx5 and Tbx3. This is the first study demonstrating gene rearrangements and their deregulated mRNA expression in radiation-transformed breast cells. Since the gene rearrangements occurred in the transformed and tumourigenic cell lines only, it is likely that these were generated in conjunction with malignant transformation of the epithelial breast cells and therefore might reflect early molecular events in breast carcinogenesis. Initial studies indicate that these gene alterations are also found in sporadic breast cancers.

KEAP1 E3 Ligase-Mediated Downregulation of NF-κB Signaling by Targeting IKKβ

IkappaB kinase beta (IKKbeta) is involved in tumor development and progression through activation of the nuclear factor (NF)-kappaB pathway. However, the molecular mechanism that regulates IKKbeta degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)-based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKbeta ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKbeta and to upregulation of NF-kappaB-derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKbeta degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKbeta ubiquitination may contribute to tumorigenesis.

Prevalence and Functional Analysis of Sequence Variants in the ATR Checkpoint Mediator Claspin

Mutational inactivation of genes controlling the DNA-damage response contributes to cancer susceptibility within families and within the general population as well as to sporadic tumorigenesis. Claspin (CLSPN) encodes a recently recognized mediator protein essential for the ATR and CHK1-dependent checkpoint elicited by replicative stress or the presence of ssDNA. Here, we describe a study to determine whether mutational disruption of CLSPN contributes to cancer susceptibility and sporadic tumorigenesis. We resequenced CLSPN from the germline of selected cancer families with a history of breast cancer (n = 25) or a multicancer phenotype (n = 46) as well as from a panel of sporadic cancer cell lines (n = 52) derived from a variety of tumor types. Eight nonsynonymous variants, including a recurrent mutation, were identified from the germline of two cancer-prone individuals and five cancer cell lines of breast, ovarian, and hematopoietic origin. None of the variants was present within population controls. In contrast, mutations were rare within genes encoding the CLSPN-interacting protein ATR and its binding partner ATRIP. One variant of CLSPN, encoding the I783S missense mutation, was defective in its ability to mediate CHK1 phosphorylation following DNA damage and was unable to rescue sensitivity to replicative stress in CLSPN-depleted cells. Taken together, these observations raise the possibility that CLSPN may encode a component of the DNA-damage response pathway that is targeted by mutations in human cancers, suggesting the need for larger population-based studies to investigate whether CLSPN variants contribute to cancer susceptibility.

Translation initiation: a critical signalling node in cancer

Mammalian target of rapamycin (mTOR) is a master regulator of translation initiation that controls the recruitment of ribosomes to mRNA templates in response to intracellular and extracellular cues. Evidence suggests that mTOR and its direct downstream targets, S6K and eIF4E/4E-BP, play significant roles in oncogenesis, and that inhibiting this pathway holds promise as an anti-proliferative approach. Recent genome-wide analyses of mutations in human cancers indicate that transformed cells activate a handful of processes and signalling pathways that are major contributors to their phenotype. Here we review the current literature implicating mTOR and translation initiation downstream of many of these various signalling pathways and processes usurped in human cancers. This review highlights the widespread activation of mTOR/eIF4E following acquisition of oncogenic lesions and its implication in promoting the transformation phenotype and indicates that targeting the control of translation initiation makes logical sense as a broad-acting therapeutic approach.

Mutations in mitochondrial DNA polymerase-gamma promote breast tumorigenesis.

Decreased mitochondrial oxidative phosphorylation (OXPHOS) is one of the hallmarks of cancer. To date the identity of nuclear gene(s) responsible for decreased OXPHOS in tumors remains unknown. It is also unclear whether mutations in nuclear gene(s) responsible for decreased OXPHOS affect tumorigenesis. Polymerase γ (POLG) is the only DNA polymerase known to function in human mitochondria. Mutations in POLG are known to cause mtDNA depletion and decreased OXPHOS resulting in mtDNA depletion syndrome (MDS) in humans. We therefore sequenced all coding exons [2-23] and flanking intron/splice junctions of POLG in breast tumors. We found that the POLG gene was mutated in 63% of the breast tumors. We identified a total of 17 mutations across the POLG gene. Mutations were found in all three domains of POLG protein, including T251I (exonuclease domain), P587L (linker region) and E1143G (polymerase domain). We identified two novel mutations that include one silent (A703A) and one missense (R628Q) mutation in the evolutionarily conserved POLG linker region. Additionally, we identified three novel mutations in the intronic region. Our study also revealed that mtDNA was depleted in breast tumors. Consistently, mutant POLG when expressed in breast cancer cells induced depletion of mtDNA, decreased mitochondrial activity, decreased mitochondrial membrane potential, increased levels of reactive oxygen species (ROS), and increased matrigel invasion. Together, our study provides the first comprehensive analysis of the POLG gene mutation in human cancer and suggests a role for POLG in 1) decreased OXPHOS in cancers and 2) in promoting tumorigenicity.

Fast simultaneous detection of K-RASmutations in colorectal cancer

BackgroundRAS genes acquire the most common somatic gain-of-function mutations in human cancer, and almost all of these mutations are located at codons 12, 13, 61, and 146.MethodsWe present a method for detecting these K-RAS hotspot mutations in 228 cases of colorectal cancer. The protocol is based on the multiplex amplification of exons 2, 3 and 4 in a single tube, followed by primer extension of the PCR products using various sizes of primers to detect base changes at codons 12, 13, 61 and 146. We compared the clinicopathological data of colorectal cancer patients with the K-RAS mutation status.ResultsK-RAS mutation occurred in 36% (83/228) of our colorectal cancer cases. Univariate analysis revealed a significant association between K-RAS mutation at codon 12 of exon 2 and poor 5-year survival (p = 0.023) and lymph node involvement (p = 0.048). Also, K-RAS mutation at codon 13 of exon 2 correlates with the size of the tumor (p = 0.03). Multivariate analysis adjusted for tumor size, histologic grade, and lymph node metastasis also indicated K-RAS mutations at codon 12 and 13 of exon 2 correlate significantly with overall survival (p = 0.002 and 0.025). No association was observed between codon 61 and 146 and clinicopathological features.ConclusionWe demonstrated a simple and fast way to identify K-RAS mutation.

Genetic Inactivation of ADAMTS15 Metalloprotease in Human Colorectal Cancer

Matrix metalloproteinases have been traditionally linked to cancer dissemination through their ability to degrade most extracellular matrix components, thus facilitating invasion and metastasis of tumor cells. However, recent functional studies have revealed that some metalloproteases, including several members of the ADAMTS family, also exhibit tumor suppressor properties. In particular, ADAMTS1, ADAMTS9, and ADAMTS18 have been found to be epigenetically silenced in malignant tumors of different sources, suggesting that they may function as tumor suppressor genes. Herein, we show that ADAMTS15 is genetically inactivated in colon cancer. We have performed a mutational analysis of the ADAMTS15 gene in human colorectal carcinomas, with the finding of four mutations in 50 primary tumors and 6 colorectal cancer cell lines. Moreover, functional in vitro and in vivo studies using HCT-116 and SW-620 colorectal cancer cells and severe combined immunodeficient mice have revealed that ADAMTS15 restrains tumor growth and invasion. Furthermore, the presence of ADAMTS15 in human colorectal cancer samples showed a negative correlation with the histopathologic differentiation grade of the corresponding tumors. Collectively, these results provide evidence that extracellular proteases, including ADAMTS15, may be targets of inactivating mutations in human cancer and further validate the concept that secreted metalloproteases may show tumor suppressor properties.

Kinome Sirna Screen Identifies SMG-1 as a Negative Regulator of Hypoxia-inducible Factor-1α in Hypoxia

Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis. To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions. This screen determined that depletion of cellular SMG-1 kinase most significantly modified cellular HIF-1 activity in hypoxia. SMG-1 is the newest and least studied member of the phosphoinositide 3-kinase-related kinase family, which consists of ATM, ATR, DNA-PKcs, mTOR, and SMG-1. We individually depleted members of the phosphoinositide 3-kinase-related kinase family, and only SMG-1 deficiency significantly augmented HIF-1 activity in hypoxia. We subsequently discovered that SMG-1 kinase activity was activated by hypoxia, and depletion of SMG-1 up-regulated MAPK activity under low oxygen. Suppressing cellular MAPK by silencing ERK1/2 or by treatment with U0126, a MAPK inhibitor, partially blocked the escalation of HIF-1 activity resulting from SMG-1 deficiency in hypoxic cells. Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1α. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1α-regulated angiogenic factor, vascular epidermal growth factor, and survival factor, carbonic anhydrase IX (CA9), as well as promoted the hypoxic cell motility. Taken together, we discovered that SMG-1 negatively regulated HIF-1α activity in hypoxia, in part through blocking MAPK activation.

Gain‐of‐function mutations and copy number increases of Notch2 in diffuse large B‐cell lymphoma

Signaling through the Notch1 receptor has a pivotal role in early thymocyte development. Gain of Notch1 function results in the development of T-cell acute lymphoblastic leukemia in a number of mouse experimental models, and activating Notch1 mutations deregulate Notch1 signaling in the majority of human T-cell acute lymphoblastic leukemias. Notch2, another member of the Notch gene family, is preferentially expressed in mature B cells and is essential for marginal zone B-cell generation. Here, we report that 5 of 63 (approximately 8%) diffuse large B-cell lymphomas, a subtype of mature B-cell lymphomas, have Notch2 mutations. These mutations lead to partial or complete deletion of the proline-, glutamic acid-, serine- and threonine-rich (PEST) domain, or a single amino acid substitution at the C-terminus of Notch2 protein. Furthermore, high-density oligonucleotide microarray analysis revealed that some diffuse large B-cell lymphoma cases also have increased copies of the mutated Notch2 allele. In the Notch activation-sensitive luciferase reporter assay in vitro, mutant Notch2 receptors show increased activity compared with wild-type Notch2. These findings implicate Notch2 gain-of-function mutations in the pathogenesis of a subset of B-cell lymphomas, and suggest broader roles for Notch gene mutations in human cancers.

GLI1 Is a Direct Transcriptional Target of EWS-FLI1 Oncoprotein

Ewing sarcoma family of tumors (ESFT) is an undifferentiated neoplasm of the bone and soft tissue. ESFT is characterized by a specific chromosomal translocation occurring between chromosome 22 and (in most cases) chromosome 11, which generates an aberrant transcription factor, EWS-FLI1. The function of EWS-FLI1 is essential for the maintenance of ESFT cell survival and tumorigenesis. The Hedgehog pathway is activated in several cancers. Oncogenic potential of the Hedgehog pathway is mediated by increasing the activity of the GLI family of transcription factors. Recent evidence suggests that EWS-FLI1 increases expression of GLI1 by an unknown mechanism. Our data from chromatin immunoprecipitation and promoter reporter studies indicated GLI1 as a direct transcriptional target of EWS-FLI1. Expression of EWS-FLI1 in non-ESFT cells increased GLI1 expression and GLI-dependent transcription. We also detected high levels of GLI1 protein in ESFT cell lines. Pharmacological inhibition of GLI1 protein function decreased proliferation and soft agar colony formation of ESFT cells. Our results establish GLI1 as a direct transcriptional target of EWS-FLI1 and suggest a potential role for GLI1 in ESFT tumorigenesis.

A TARBP2 mutation in human cancer impairs microRNA processing and DICER1 function

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by targeting messenger RNA (mRNA) transcripts. Recently, a miRNA expression profile of human tumors has been characterized by an overall miRNA downregulation. Explanations for this observation include a failure of miRNA post-transcriptional regulation, transcriptional silencing associated with hypermethylation of CpG island promoters and miRNA transcriptional repression by oncogenic factors. Another possibility is that the enzymes and cofactors involved in miRNA processing pathways may themselves be targets of genetic disruption, further enhancing cellular transformation. However, no loss-of-function genetic alterations in the genes encoding these proteins have been reported. Here we have identified truncating mutations in TARBP2 (TAR RNA-binding protein 2), encoding an integral component of a DICER1-containing complex, in sporadic and hereditary carcinomas with microsatellite instability. The presence of TARBP2 frameshift mutations causes diminished TRBP protein expression and a defect in the processing of miRNAs. The reintroduction of TRBP in the deficient cells restores the efficient production of miRNAs and inhibits tumor growth. Most important, the TRBP impairment is associated with a destabilization of the DICER1 protein. These results provide, for a subset of human tumors, an explanation for the observed defects in the expression of mature miRNAs.

From Concept to Reality: The Long Road to c-Met and RON Receptor Tyrosine Kinase Inhibitors for the Treatment of Cancer

c-Met and RON are receptor tyrosine kinases (RTK) that are closely related, both from a homology as well as from a functional stand point. Both receptors can induce cell migration, invasion, proliferation and survival in response to their respective ligand. Moreover, both possess oncogenic activity in vitro, in animal models in vivo and are often deregulated in human cancers. c-Met attracted a lot of interest shortly after its discovery in the mid-1980s because of its unusual role in cell motility. Moreover, a causal role for c-Met activating mutations in human cancer propelled an intensive drug discovery effort throughout the research and pharmaceutical communities to find inhibitors of c-Met. While c-Met is now a well-accepted target for an anti-cancer drug, less is known about the role of RON in cancer. Interestingly, despite their many common attributes, c-Met and RON are activated by different mechanisms in cancer cells. Because of the homology between the two RTKs, some small molecule kinase inhibitors of c-Met have inhibitory activity on RON, opening the door to exploring the role of both receptors in human cancers. In this review we will discuss the relevance of both c-Met and RON deregulation in human cancers and the progress so far in identifying small molecule kinase inhibitors that can block the activity of these targets in vitro and lead to anti-tumor effects in animal models.

PTEN: a new guardian of the genome

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is a phosphatase that antagonizes the phosphoinositol-3-kinase/AKT signaling pathway and suppresses cell survival as well as cell proliferation. PTEN is the second most frequently mutated gene in human cancer after p53. Germline mutations of PTEN have been found in cancer susceptibility syndromes, such as Cowden syndrome, in which over 80% of patients have mutations of PTEN. Homozygous deletion of Pten causes embryonic lethality, suggesting that PTEN is essential for embryonic development. Mice heterozygous for Pten develop spontaneous tumors in a variety of organs comparable with the spectrum of its mutations in human cancer. The mechanisms of PTEN functions in tumor suppression are currently under intense investigation. Recent studies demonstrate that PTEN plays an essential role in the maintenance of chromosomal stability and that loss of PTEN leads to massive alterations of chromosomes. The tumor suppressor p53 is known as a guardian of the genome that mediates the cellular response to environmental stress, leading to cell cycle arrest or cell death. Through completely different mechanisms, PTEN also protects the genome from instability. Thus, we propose that PTEN is a new guardian of the genome. In this review, we will discuss new discoveries on the role of PTEN in tumor suppression and explore mechanisms by which PTEN maintains genomic stability.

Bcl-xL and UVRAG Cause a Monomer-Dimer Switch in Beclin1

Beclin1 has a key regulatory role in the initiation of autophagy and is a tumor suppressor. We have examined the interplay between viral or human Bcl-2-like proteins and UVRAG and their opposite effects on Beclin1. We show that Beclin1 forms a dimer in solution via its coiled-coil domain both in vivo and in vitro. Viral Bcl-2 binds independently to two sites on the Beclin1 dimer, one with high affinity and one with lower affinity, whereas human Bcl-x(L) binds both sites equally with relatively low affinity. UVRAG disrupts the Beclin1-dimer interface, forming a heterodimer with Beclin1, suggesting that this is how UVRAG causes its effects on Beclin1 to activate autophagy. Both Bcl-2-like proteins reduce the affinity of UVRAG for Beclin1 approximately 4-fold, suggesting that they stabilize the Beclin1 dimer. Moreover, coimmunoprecipitation assays show that UVRAG substantially reduces Beclin1 dimerization in vivo. These data explain the concentration-dependent interplay between Bcl-2, UVRAG, and Beclin1, as both tumor suppressors, UVRAG and Beclin1, have single-copy mutations in human cancers. Furthermore, our data suggest that an alternative strategy for developing anti-cancer compounds would be to disrupt the Beclin1-dimer interface.

Advances in chemical carcinogenesis: a historical review and prospective.

The American Association for Cancer Research has been the citadel for communicating research on chemical carcinogens for over a century. It therefore seems appropriate that a review of chemical carcinogenesis inaugurates a series of articles highlighting advances in understanding, treating, and

Design and analysis issues in genome-wide somatic mutation studies of cancer

The availability of the human genome sequence and progress in sequencing and bioinformatic technologies have enabled genome-wide investigation of somatic mutations in human cancers. This article briefly reviews challenges arising in the statistical analysis of mutational data of this kind. A first challenge is that of designing studies that efficiently allocate sequencing resources. We show that this can be addressed by two-stage designs and demonstrate via simulations that even relatively small studies can produce lists of candidate cancer genes that are highly informative for future research efforts. A second challenge is to distinguish mutated genes that are selected for by cancer (drivers) from mutated genes that have no role in the development of cancer and simply happened to mutate (passengers). We suggest that this question is best approached as a classification problem and discuss some of the difficulties of more traditional testing-based approaches. A third challenge is to identify biologic processes affected by the driver genes. This can be pursued by gene set analyses. These can reliably identify functional groups and pathways that are enriched for mutated genes even when the individual genes involved in those pathways or sets are not mutated at sufficient frequencies to provide conclusive evidence as drivers.