Research Article

Colon Tumors with the Simultaneous Induction ofDriver Mutations in APC, KRAS, and PIK3CA StillProgress through the Adenoma-to-carcinomaSequenceJamieN. Hadac1, AlyssaA. Leystra1,Terrah J. PaulOlson2,Molly E.Maher3, SusanN. Payne4,Alexander E. Yueh3, Alexander R. Schwartz5, Dawn M. Albrecht5, Linda Clipson1,CheriA.Pasch4,KristinaA.Matkowskyj4,6,7,RichardB.Halberg4,5,and DustinA.Deming3,4,7

Abstract

Human colorectal cancers often possess multiple mutations,including three to six driver mutations per tumor. The timing ofwhen these mutations occur during tumor development andprogression continues to be debated.More advanced lesions carrya greater number of driver mutations, indicating that colontumors might progress from adenomas to carcinomas throughthe stepwise accumulation of mutations following tumor initia-tion. However, mutations that have been implicated in tumorprogression have been identified in normal-appearing epithelialcells of the colon, leaving the possibility that these mutationsmight be present before the initiation of tumorigenesis. Weutilized mouse models of colon cancer to investigate whethertumorigenesis still occurs through the adenoma-to-carcinoma

sequence when multiple mutations are present at the time oftumor initiation. To create a model in which tumors couldconcomitantly possess mutations in Apc, Kras, and Pik3ca, wedeveloped a novelminimally invasive technique to administer anadenovirus expressing Cre recombinase to a focal region of thecolon. Here, we demonstrate that the presence of these additionaldriver mutations at the time of tumor initiation results inincreased tumor multiplicity and an increased rate of progressionto invasive adenocarcinomas. These cancers can even metastasizeto retroperitoneal lymph nodes or the liver. However, despitehaving asmany as three concomitant driver mutations at the timeof initiation, these tumors still proceed through the adenoma-to-carcinoma sequence. Cancer Prev Res; 8(10); 952–61. �2015 AACR.

IntroductionColorectal cancer is the second-leading cause of cancer-related

mortality in theUnited States (1) . An improved understanding ofthe processes by which tumorigenesis occurs will allow for therational development of chemopreventive and therapeutic agents.The canonical adenoma-to-carcinoma sequence has been pro-posed to describe the processes by which mutations in drivergenes accumulate over time causing the progression of adenomasto invasive adenocarcinomas in the colon (2, 3).

An important early step in tumorigenesis is the acquisition ofalterations in the Adenomatous Polyposis Coli (APC) tumor sup-pressor gene. Loss of this gatekeeper gene is thought to be theinitiating event in the majority of sporadic human colorectalcancers with approximately 80% to 90% of human coloncancers harboring somatic mutations in APC (4–6). In thecanonical sequence, tumor initiation caused by loss of APC isfollowed by mutations in other genes including KRAS, TP53,PIK3CA, and BRAF (7). Some studies have questioned whetherthe accumulation of mutations over time is necessary for coloncancer development (8–11). For example, KRAS mutationshave been detected in non-neoplastic tissue of the colon(8, 12). This observation indicates that somatic mutationsoccurring before tumor initiation might subsequently influencetumor development and progression (13). In addition, it couldbe a potential explanation for interval cancers that formbetween routine screening colonoscopies.

Because of the prevalence of APC loss in human colorectalcancer,mice carryingmutations inApc, themurine homolog, havebeen generated tomore fully understand intestinal tumorigenesisand investigate pharmacologic strategies for chemoprevention(14–16). Here, we utilized genetically engineered mouse modelsof colon cancer to examine the adenoma-to-carcinoma sequencein the setting of the concurrent induction of common drivermutations. We developed a minimally invasive procedure forinoculating an adenovirus expressing the Cre recombinase(Adeno-Cre) to induce multiple mutations at a desired location

1Department of Oncology, University of Wisconsin, Madison,Wiscon-sin. 2Division of General Surgery, Department of Surgery, University ofWisconsin, Madison,Wisconsin. 3Division of Hematology and Oncol-ogy, University of Wisconsin, Madison,Wisconsin. 4University of Wis-consin Carbone Cancer Center, University of Wisconsin, Madison,Wisconsin. 5Division of Gastroenterology and Hepatology, Depart-ment of Medicine, University of Wisconsin, Madison, Wisconsin.6Department of Pathology and Laboratory Medicine, University ofWisconsin, Madison,Wisconsin. 7WilliamSMiddletonMemorial Veter-ans Hospital, Madison,Wisconsin.

Note: Supplementary data for this article are available at Cancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: Dustin A. Deming, University of Wisconsin, 600 High-land Ave., K6/544, Madison, WI 53792. Phone: 608-265-1042; E-mail:ddeming@medicine.wisc.edu

doi: 10.1158/1940-6207.CAPR-15-0003

�2015 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 8(10) October 2015952

in the colon. This method allows for tumors to be initiated with apredetermined mutation profile at a specific time and locationdesired by the investigator.

Materials and MethodsMouse husbandry

All animal studies were conducted under protocols approvedby the Institutional Animal Care and Use Committee at theUniversity of Wisconsin-Madison (Madison, WI), followingthe guidelines of the American Association for the Assessmentand Accreditation of Laboratory Animal Care. FCmice (FVB/N-Tg(Fabp1-Cre)1Jig/Nci; NCI Mouse Repository, strain number01XD8), KrasG12D/þ mice (C57BL/6N.Cg-Krastm4Tyj/CjDswJ;The Jackson Laboratory, stock number 019104), Pik3cap110

�mice

(C57BL/6-Gt(ROSA)26Sortm7(Pik3cap110�,EGFP)Rsky/J; The JacksonLaboratory, stock number 012343), Apcfl/fl mice (C57BL/6.Cg-Apctm2Rak/Nci; NCIMouseRepository, strain number 01XAA),and mT/mG mice (B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-

EGFP)Luo/J; The Jackson Laboratory, stock number 007676) weremaintained and genotyped as previously described (17–21). ForFC and mT/mG, a 1 denotes carrier and 0 noncarrier. Pik3cap110

�

and KrasG12D/þ mice were also crossed to Apcfl/fl mice to generateApcfl/fl KrasG12D/þ, Apcfl/fl Pik3cap110

�, and Apcfl/fl KrasG12D/þ

Pik3cap110�mice on a homogeneous B6 genetic background for

Adeno-Cre delivery. In addition, mT/mG1 mice were crossed togenerate B6 mT/mG1 Apcfl/fl KrasG12D/þ Pik3cap110

�mice. All

Pik3cap110�mice were hemizygous for this allele.

Nonsurgical exposure of the colon to Adeno-CrePolyethylene tubing (I.D. 1.4 mm, O.D. 1.90 mm; Becton

Dickinson, Sparks, MD) was cut to sizes appropriate for mice(7–12 cm). A 1-cm window was notched into the tubing and theend of the tubing was closed with edges being rounded to avoidperforation of the bowel (Fig. 1A). Marks corresponding to 1 cmintervals were made on the tubing. A longitudinal stripe was alsoapplied corresponding to theorientationof thewindow. Similarlysized polyethylene tubing was cut to size without a slot to be usedas a sheath for a 2.2-mm caliber soft bristle brush (DenTek OralCare).

Mice were anesthetized using 2% isoflurane. The colon wasirrigated with PBS. A narrow ribbon of GelFoam (Pharmacia andUpjohn) was inserted into the window cut into the polyethylenetube. Of note, 200 mL of 0.05% trypsin (Hyclone) was injectedinto the tubing and inserted into the mouse colon at the desireddepth and radial orientation. After 10 minutes, the slotted tubewas removed and the sheath with the small soft bristle brush wasintroduced at the same intraluminal location. The brush was thenused to abrade the epithelium for up to 3 minutes. After PBSirrigation, a slotted tubing containing GelFoam was then filledwith 200 mL PBS containing 109 PFU of Adeno-Cre (Ad5CMVCreand Ad5CMVEmpty, University of Iowa Gene Transfer VectorCore, IA). After 30 minutes of incubation, the tubing wasremoved. Owing to the anatomical limitations of the mouse,only themost distal half of the colon (�4 cm) could be inoculatedin thisway.Mice recoveredquickly after the procedure anddid notexhibit overt signs of pain or distress following the procedure, asthey quickly became active. Note that nonsteroidal anti-inflam-matory drugs were not used for post-procedure analgesia as theseagents have been shown to suppress intestinal tumorigenesis inboth humans and laboratory mice.

3. Brush colon – 2 minutes

4. Adeno-Cre – 30-minute incubation

2. Trypsin – 10-minute incubation

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Figure 1.A minimally invasive technique for inoculation of the colon with Adeno-Creinitiates tumorigenesis in Apcfl/flmice. We have developed a novel techniquethat can be utilized to sequester Adeno-Cre within the colon withoutlaparotomy, as was required with prior methodologies. A, in this technique, aslotted tube housing GelFoam allows for the treatment of the colon withtrypsin. Abrasion of the colon is then performed before incubation withAdeno-cre–soaked GelFoam at the desired location. B, colonic tumors arevisualized by endoscopy in 3 to 7 weeks following treatment with Adeno-Creandmonitored over timewith serial endoscopy. C, interestingly, these tumorshave varying growth patterns over 14 weeks of observation, despite a similarinitiation. D, histologic evaluation demonstrated that the majority (83%) ofthese lesionswere adenomaswith nuclear CTNNB1 consistent with activationof theWNTpathway. The area indicated by the rectangle is shownenlarged atthe right; at the far right the same area in an adjacent slide stained for CTNNB1is shown. Size bar ¼ 1 mm.

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Murine colonoscopy/PET imagingMice were anesthetized using 2% isoflurane and the colons

were flushedwith PBS. The Coloview Systemwas used tomonitortumor formation and growth in the distal half of the colon aspreviously described (Karl Storz; ref. 22). ImageJ analysis wasutilized to measure the percent lumen occlusion as previouslydescribed (23).

Animals were fasted for at least 6 hours before injection of18F-FDG (160 mCi; IBA Molecular). After injection, the animalswere kept under anesthesia for 60 minutes and then preparedfor microPET colonography as described previously (24).

Histology and IHCMice were euthanized and the colons were excised and opened

longitudinally. Tumor tissue was fixed in 10% buffered formalin.Fixed tumors were embedded in paraffin and cut into 5 mmsections. Every tenth section was stained with hematoxylin andeosin (H&E) for histologic review. IHC was carried out asdescribed previously (25) with the exception that tissues wereblocked with Background Sniper (Biocare Medical) for 10 min-utes. The primary antibodies included rabbit anti-phospho-AKT(Ser473, 1:100, Cell Signaling Technology #4060), rabbit anti-phospho-S6 Ribosomal Protein (Ser235/236, 1:50, Cell SignalingTechnology #4858), rabbit anti-CTNNB1 (1:100–1:200, CellSignaling Technology #8480), rabbit anti-Ki67 (1:400, Cell Sig-naling Technology #12202), and rabbit anti-phospho-ERK1/2(1:400, Cell Signaling Technology #4370). The Ki-67 prolifera-tion index was measured as the percent of nuclei staining positivefor Ki-67 per tumor using ImmunoRatio, an ImageJ plugin(http://jvsmicroscope.uta.fi/sites/default/files/software/immu-noratio-plugin/index.html).

Recombination testingTo validate that all alleles were recombined within tumors,

neoplastic tissuewas scraped from FFPE sections with a sterile size10 surgical blade with normal epithelium left behind. DNA wasisolated from the scraped tissue using theMaxwell 16 FFPE TissueLEV DNA Purification Kit (Promega). Samples with a sufficientamount of DNA were analyzed for Cre-mediated recombinationbetween loxP sites.

Recombination of the loxP sites within the Apcfl allele andupstream of the KRASG12D allele was confirmed using previ-ously described PCR primers and protocols (http://jacks-lab.mit.edu/protocols/genotyping/kras_cond; ref. 20). Recombina-tion of the loxP sites upstream of the Pik3cap110

�allele was

confirmed using the forward primer 50-CGCGGTTGAGGA-CAAACTCT-30 and the reverse primer 50-ACCATAATTCCAC-CACCACCA-30. Cycling conditions were one cycle of 94�C for3 minutes, followed by 35 cycles of 94�C for 20 seconds, 61�Cfor 60 seconds, and 72�C for 60 seconds, with one finalextension cycle at 72�C for 2 minutes. Recombined DNAresulted in amplification of a 561-bp product.

ResultsColon tumors can be initiated using a noninvasive inoculationof the Adeno-Cre virus

KRAS and PIK3CAmutations have been identified as occurringconcomitantly with the loss of APC in human adenomas andcarcinomas (26, 27). Multiple investigations have examinedmurine models with combinations of loss of APC and activating

mutations in either KRAS or PIK3CA (28–31). These studies hadlimitations. C57BL/6 (B6) mice carrying the Min allele of Apc(ApcMin/þ) have a ubiquitous spatial and temporal expression of amutant germline allele resulting in alterations of homeostasisalong the entire intestinal tract (14). The life expectancy of thesemice is limited due to their high tumor burden, especially of thesmall intestine, resulting in anemia or intestinal obstruction. B6ApcMin/þ mice do not develop advanced colon cancers as aconsequence of this shortened lifespan. Finally, triggering mul-tiple mutations in the colon concurrently by expressing Crerecombinase from the FABP-1 promoter is lethal (SupplementaryFig. S1). Todeterminewhether additional concomitantmutationswould alter the progression of colon tumors through the adeno-ma-to-carcinoma sequence alternative methodologies wererequired. Amodel allowing for spatial control of tumor initiationthat can be used to express a number of mutations was needed tofurther explore their effects on tumorigenesis.

Although there are multiple methods of expressing Cre in themouse, including organ-specific promoter-based systems, thedelivery of an adenovirus carrying Cre recombinase allows forthe greatest spatio-temporal control of initiation of colon tumors(18). To sequester the virus in the colon, an invasive laparotomyprocedure has been described (23). Surgical clips were directlyplaced both proximally and distally around the colon afterexposure of the peritoneal cavity. Adeno-Cre was then injectedinto the area between the clips. This surgical Adeno-Cre instilla-tion technique allows for the development of colon tumorswithout the comorbidities related to Apc mutations occurringoutside of the area of interest. However, this surgical interventionis no trivial task; preparation, surgery, and recovery may allcontribute detrimental effects to the overall health of the animal.Unintended consequences from surgical wounds may alter var-ious cellular mechanisms, including inflammatory and wound-healing responses, involved in tumorigenesis (32).

To overcome limitations of the surgical approach, we devel-oped adevice to nonsurgically inoculate Adeno-Cre into the colonin homozygous Apcfl/fl mice (Fig. 1A; ref. 20). Upon expression ofCre recombinase in thesemice, exon 14 ofApc is excised, resultingin the expression of a truncated, non-functional APC protein. Creexpression controls the location and time at which tumors form,in turn alleviatingmany of the limitations associatedwithmodelscarrying germline Apc mutations. This nonsurgical approachinduced tumorigenesis in a physiologically relevant setting, whileminimizing the systemic effects of an operative procedure.

Biocompatible materials have been used to enhance the deliv-ery of viruses in a variety of research settings (33). One suchbiomaterial, GelFoam (Pharmacia and Upjohn), is a collagen-based sponge. It has been used to deliver viruses in gene therapyresearch due to its ability to localize and protect virus (34).GelFoam saturated in adenovirus solution has been implantedin mice and rats to facilitate gene expression for the studyof wound healing and to elicit immune responses in tumors(35, 36). These studies have demonstrated that GelFoam can beused to improve the sustained delivery of adenovirus in vivo.Therefore, we found it an attractive alternative to surgery for usein localizing the virus in specific areas of the mouse.

A slotted tube housing GelFoam soaked with Adeno-Cre wasplaced 1.5 to 3.5 cm into the colon of each prepared Apcfl/flmouse(see Materials and Methods). After the procedure, tumor forma-tion and growth were monitored by endoscopy. Tumors werevisible as early as 3 weeks post-treatment and these tumors were

Hadac et al.

Cancer Prev Res; 8(10) October 2015 Cancer Prevention Research954

monitored over 14 weeks post-treatment. Fifty-eight percent(18/31) of treated Apcfl/fl mice developed tumors. Tumorsoriginated at the desired location, both in radial orientationand depth in the colon (Fig. 1B). No tumors developed in miceinfected with adenovirus carrying an empty vector.

Differential growth patterns occur in tumors after thesimultaneous loss of both normal APC alleles

Small animal endoscopy was performed every other week overa 14-week period to follow tumor growth in homozygous Apcfl/fl

mice treated with Adeno-Cre. Tumors visible one month afteradenoviral treatment were relatively small, occluding less than30% of the lumen. However, in the following weeks, tumorsexhibited many different patterns of growth (Fig. 1C). Sometumors grew rapidly to eventually occlude the entire lumen, whileothers grew more incrementally, or even remained stable in sizeover this time. One small tumor ultimately regressed. Thesedynamic growth patterns, observed even in the earliest adenomas,were similar to tumor growth patterns in other murine colontumor models with loss of APC (25).

After 14 weeks of monitoring, necropsy was performed. Noneof the mice became moribund before this time point. Histologicevaluation was completed on six tumors which revealed that 83%were adenomas, and 17% were invasive adenocarcinomas withinvasion into the muscularis mucosa (Fig. 1D). No evidence ofregional nodal disease or metastatic disease was observed.

Epithelial tumors arise secondary to inoculation of the colonwith Adeno-Cre

When the Adeno-Cre virus is instilled into the colon, it ispossible that the virus is infecting cells other than the epithelialcells lining the intestine. To make certain that only epithelial cellswere being infected with the Adeno-Cre virus, mT/mG1 Apcfl/fl

KrasG12D/þ Pik3cap110�mice were treated with Adeno-Cre. mT/mG

is a reporter from which red (tdTomato) fluorescent protein isubiquitously expressed preceding Cre recombinase expressionand GFP is expressed following Cre recombinase expression.Green fluorescence was detected only within the colonic epithe-lium confirming that the intended cell type was being infectedwith the Adeno-Cre (Fig. 2). Nuclear localization of CTNNB1(b-catenin) and activation of the anticipated downstream signal-ing cascades were observed within the transformed epithelial cellsindicating that recombination following Cre expression wasoccurring as anticipated (Fig. 2).

Adeno-Cre tumor initiation rate depends upon the mutationprofile

Our minimally invasive technique allows for temporaland spatial control of the initiation of colon tumors with mul-tiple simultaneousmutations.MicewithApcfl/fl,KrasG12D/þ, and/or Pik3cap110

�were generated and treated with Adeno-Cre

(Fig. 3A). After 3 to 4 weeks, tumors were identified on endos-copy. When additional driver mutations were present in addi-tion to the loss of APC, an increase in the rate of tumordevelopment was observed. Tumors developed in 79% ofApcfl/fl mice carrying KrasG12D and/or Pik3cap110

�compared with

58% of Apcfl/fl mice without these additional driver mutations[Fig. 3A, P (one-sided) ¼ 0.031, Barnard exact test]. All micewere treated according to the same Adeno-Cre protocol and onthe same homogeneous genetic background. In the 14 samplesthat were successfully tested, recombination was confirmed innearly all cases (12/14, Supplementary Fig. S2). Exceptions

Figure 2.Multiple mutations were simultaneously expressed within the epithelium ofthe colon following a novel minimally invasive Adeno-Cre technique resultingin the development of colon tumors. To be certain that our mutations werebeing activated only in the epithelial cells of the colon with this technique, weutilized mT/mG1 Apcfl/fl Pik3cap110� mice. Upon Cre recombination, a geneencoding a red fluorescent marker in these mice is excised and a greenfluorescent protein is expressed. A representative histologic section isdisplayed demonstrating that Cre-mediated recombination has onlyoccurred in the epithelial cells of these lesions. IHC confirmed CTNNB1(b-catenin) localization to the nucleus and activation of the PI3K signalingcascade with intense staining for phosphorylated AKT (pAKT) andphosphorylated RPS6 (pRPS6), as expected. Activation of ERK1/2 was notobserved in these tumors. The area indicated by each rectangle is shownenlarged at the right. Size bar ¼ 1 mm.

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were found within two of three samples from a single Apcfl/fl

KrasG12D/þ Pik3cap110�mouse: only two of the three Cre-depen-

dent alleles appeared to recombine in two tumors, but all threeCre-dependent alleles recombined in the third tumor.

In the setting of multiple mutations, Adeno-Cre inductionresulted in adenoma formation

Mice with Apcfl/fl plus KrasG12D/þ and/or Pik3cap110�were gen-

erated and treated with Adeno-Cre to determine how the simul-taneous induction of these mutations would alter tumor biology.Tumors were identified 3 to 8weeks after viral treatment (Fig. 3B).Histologic sectioning of tumors revealed that all tumors fromthese mice carrying mutations in two to three genes at this earlytime point were small adenomas. An example of a small adenomafrom an Apcfl/fl Pik3cap110

�mouse is presented in Fig. 3B. The

expected nuclear localization of CTNNB1 and activation of thePI3K cascade with abundant phosphorylation of RPS6 wereobserved (Fig. 3B).

The addition of Kras and Pik3ca mutations does notsignificantly alter the rate of proliferation in colon tumorscompared with loss of APC alone

To estimate the rate at which the tumors with different muta-tion profiles were growing, Apcfl/fl Pik3cap110

�mice and Apcfl/fl

KrasG12D/þPik3cap110�miceweremonitoredwith serial endoscopy

every 2 weeks following treatment with Adeno-Cre (Fig. 4A).Lumen occlusion by the colonic tumor was used as a marker ofcellular proliferation within tumors. Occlusion of 75% of thecolonic lumen was observed in 65% of tumors in Apcfl/fl

Pik3cap110�, andApcfl/flKrasG12D/þ Pik3cap110

�mice. For the tumors

that eventually occluded at least 75%of the lumen, themean timerequired to reach 75% occlusion was 15 weeks. No statisticallysignificant difference in the mean time to 75% lumen occlusionwas observed between tumorswith APC loss and the addition of 1or 2 oncogenes (Fig. 4B). Interestingly, in one instance, a tumorfrom an Apcfl/fl Pik3cap110

�mouse regressed (Supplementary

Fig. S3). Histologic analysis of this regressed area revealed apredominant lymphocytic infiltrate, indicating a potentialimmune-mediated mechanism for the regulation of some colontumors.

To confirm that mean time to lumen occlusion as is a usefulmarker for cellular proliferation within tumors, histologic sec-tioning of tumors from Apcfl/fl, Apcfl/fl Pik3cap110

�, and Apcfl/fl

KrasG12D/þ Pik3cap110�mice was performed and these sections

were stained for Ki-67, a standard marker of cell proliferation(Fig. 4C). The Ki-67 proliferative index (Ki-67 PI) was measuredas the percentage of nuclei staining positive for Ki-67 per tumor.The mean Ki-67 PI of Apcfl/fl tumors was 11.1% (range 0.8–24.8),Apcfl/fl Pik3cap110

�tumors was 18.5% (range 7.3–25.8), and Apcfl/fl

KrasG12D/þ Pik3cap110�was 15.7% (range 11.3–20.8). A trend for

increased Ki-67 PI was observed in the presence of additionalmutations, but this was not statistically significant (P ¼ 0.22).

Tumors with simultaneous loss of Apc and oncogenicmutations in Kras and Pik3ca progress from adenomas toadenocarcinomas to metastatic disease

Additional Apcfl/fl Pik3cap110�and Apcfl/fl KrasG12D/þ Pik3cap110

�

mice were monitored with serial endoscopies (SupplementaryTable S1). After 14 weeks, subsets of these mice underwentnecropsy. At this time-point, approximately 50% of tumors fromeach these genetic profiles were invasive adenocarcinomas. The

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Figure 3.Apcfl/fl KrasG12D/þ(A2K1P0), Apcfl/fl Pik3cap110

�(A2K0P1), and Apcfl/fl KrasG12D/þ

Pik3cap110�(A2K1P1) mice were treated with Adeno-Cre at 40 to 50 days of

age. A, incidence of tumors increases with the addition of KrasG12D/þand/orPik3cap110

�to the mutation profile [due to the relatively small sample sizes

and the similar tumor incidence rates amongA2K1P0,A2K0P1, andA2K1P1mice,the tumor incidence data from these strains were pooled (31/39) forcomparisonwithA2K0P0 (18/31); � ,P (one-sided)¼0.031, Barnard exact test].B, tumors were identified by endoscopy in the colon as early as 3 to 4weeks after viral inoculation. Histologic sectioning was performed on tumorsshortly after identification on endoscopy revealing that these lesions weresmall adenomas without evidence of invasion into the muscularis mucosa.An adenoma from an Apcfl/fl Pik3cap110

�mouse is shown here. Nuclear

localization of CTNNB1 and phosphorylation of RPS6 were observed withoutphosphorylation of ERK1/2 indicating that recombination was occurring asexpected. The area indicated by each rectangle is shown enlarged at the right.Size bar ¼ 500 mm.

Hadac et al.

Cancer Prev Res; 8(10) October 2015 Cancer Prevention Research956

remaining tumors were adenomas with many of them exhibitinghigh-grade dysplasia.

The remaining Apcfl/fl Pik3cap110�

and Apcfl/fl KrasG12D/þ

Pik3cap110�mice were aged until moribund. No difference in

survival was observed between the two groups with a mediansurvival of approximately 200 days (Fig. 5A). The tumorsprogressed from small polypoid lesions to invasive adenocar-cinomas that filled the majority of the colonic lumen (Fig. 5B).These tumors even progressed to "apple-core" lesions oftenseen in advanced colon cancers in humans (Fig. 5B, far right;ref. 37). When these mice were moribund, only 20% of theirtumors were preinvasive lesions, all of which were associatedwith high-grade dysplasia. The remaining 80% of tumors inthese mice were invasive adenocarcinomas, many of which hada significant proportion of the tumor extending beyond themuscularis propria to involve the serosa (Fig. 5C). Metastaticdisease was identified in two of these mice, including metas-tases to a retroperitoneal para-aortic lymph node (Fig. 6A) andmetastatic cancer within the liver (Fig. 6B). These are commonlocations for human colorectal cancer to metastasize. No lungmetastases were identified.

DiscussionThe progression froman adenoma to an invasive carcinoma is a

histologic determination defined by spread of tumor into thesubmucosa. Invasion into the submucosa has been associatedwith the presence of additional driver mutations beyond the lossof APC, but does not necessitate molecular progression beyondmutations that have been observed in adenomas. An importantconsideration, often ignored, about colon cancer tumorigenesis isthe time necessary for the acquired mutations to alter the phe-notype of the tumor. This is important when investigating thepotential timing of acquired mutations.

Activation of the WNT signaling cascade following the loss ofAPC or mutations in CTNNB1 results in tumorigenesis in themajority of human colorectal cancers (38, 39). Although activa-tion of the WNT pathway is important for tumor initiation, it isnot sufficient for progression to an invasive cancer (40). In fact,the vast majority of polyps that form due to loss of APC will notbecome invasive cancers and some will actually regress (41).Additional genetic alterations including KRAS, BRAF, PIK3CA,and TP53mutations are important for adenomas to develop high-grade dysplasia and progress to invasive adenocarcinomas (42).The timing of when these additional genetic changes occur hasbeen debated (43), though these additional genetic changes likelyoccur very early in tumorigenesis.

Because of the proliferative nature of the intestine, mutationsare known to accumulate over time in the normal epithelium(13). In colon cancers, an average of approximately 90mutationsare seen per tumor (44). This number changes significantlydepending upon the age at which the patient develops cancer.Most of thesemutations are considered to be passengermutationsand are not thought to engender a selective growth advantage(45). It has been estimated that half, if not more, of thesemutations occur before tumor initiation (13). To investigatewhether driver mutations can occur before tumor initiation,investigations into the presence of KRASmutations in the normalepithelium have been performed. KRAS mutations have beenidentified in colon cancers, adenomas, tumor-associated epithe-lial tissue, and completely normal appearing mucosa (46). This

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Figure 4.TheadditionofKrasandPik3camutations to the lossofAPChasamodesteffecton tumor proliferation. A, Apcfl/fl (A2K0P0), Apcfl/fl Pik3cap110

�(A2K0P1), and

Apcfl/fl KrasG12D/þ Pik3cap110�(A2K1P1) mice were treated with Adeno-Cre and

followedwith serial endoscopy. B, change in percent lumen occlusionwas usedas amarker of tumor proliferation. The time required for tumors to occlude 75%of the lumen was determined. No difference was seen between groups. C, toconfirm these results these tumorswere sectionedandstained forKi-67.TheKi-67 proliferation index was calculated for each tumor. D, no statisticallysignificant increase in Ki-67 staining was observed when additional mutationsoccurred concomitantly with loss of APC (P ¼ 0.35, Kruskal–Wallis test). Thearea indicatedbyeach rectangle is shownenlargedat the right. Sizebar¼ 1mm.

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indicates that cells with mutations that are important for tumorprogression, such as KRAS, might be present before tumorinitiation.

If driver mutations are present within intestinal epithelial cellsbefore or at least at the time of tumor initiation, tumor biologymay be dramatically affected. Here, we show that the presence ofKRAS and PIK3CA mutations at the time of initiation owing toloss of APC results in increased tumor multiplicity and anincreased rate of progression to invasive adenocarcinomas. Thetumors in the models described here even metastasize to

Figure 6.Metastatic colon adenocarcinoma was identified within Adeno-Cre treatedmice. An Apcfl/fl KrasG12D/þ Pik3cap110

�mouse became moribund after

treatment with Adeno-Cre. A, 18F-FDG PET imaging demonstrated an avidpara-arotic lymph node in addition to increased avidity of the primarytumor. Enlarged adenopathy was identified at necropsy. On histologicsectioning, well-differentiated adenocarcinoma consistent with thecolonic primary tumor was observed. Staining for CTNNB1 demonstratednuclear localization consistent with loss of APC. B, in addition, metastaticadenocarcinoma in the liver was identified in a moribund Adeno-Cre–treated Apcfl/fl Pik3cap110

�mouse. A hepatic mass was observed grossly.

H&E staining of histologic sections demonstrated a well-differentiatedadenocarcinoma arising within the liver. Staining demonstrated nuclearlocalization of CTNNB1 consistent with the colonic primary tumor. Thearea indicated by each rectangle in photos of stained sections is shownenlarged at the right. Size bars, 500 mm.

0

25

50

75

100

0 100 200 300 400

Surv

ival

(%)

Days of age

A2K0P1

A2K1P1

A

B

C

CTN

NB

1H

&E

pAK

TpE

RK

1/2

Figure 5.Tumors with multiple driver mutations present at the time of initiationprogressed through the adenoma to carcinoma sequence. Apcfl/fl Pik3cap110

�

(A2K0P1) and Apcfl/fl KrasG12D/þ Pik3cap110�(A2K1P1) mice were treated with

Adeno-Cre, as described, at 40 to 50 days of age and aged until moribund. A,nodifference in survival between these twogroupswas noted. B, the resultingtumors were followed with serial endoscopy; they developed initially aspolypoid lesions within the colon and progressed to occlude the majority ofthe lumen. These tumors could be followed as long as 12 months after viralinoculation and developed into "apple-core" lesions, as seen in the far rightphoto, similar to advanced colorectal tumors in humans. These mice becamemoribund due to anemia or intestinal obstruction. At necropsy, large colontumors approached 1 cm in diameter. C, upon histologic analysis, theselesions were invasive adenocarcinomas penetrating through muscularispropria and into the serosa inmany instances. H&E staining of a large invasivecancer from anApcfl/flKrasG12D/þPik3cap110

�(A2K1P1)mouse is shown. Nuclear

localization of CTNNB1 and activation of AKT and ERK1/2 signaling wereobserved. The area indicated by each rectangle shown enlarged at the right.Scale bar ¼ 1 mm.

Cancer Prev Res; 8(10) October 2015 Cancer Prevention Research958

Hadac et al.

retroperitoneal lymph nodes and the liver at relatively shortintervals, indicating the potential for an increased probability ofearly metastatic spread. These results could explain interval can-cers that develop in patients that are routinely screened followingcurrent recommendations. Of note, the results from these experi-ments might not be directly translated for all mutation profiles,and it is possible that certain mutations might be able to circum-vent the adenoma-to-carcinoma sequence and need to be studiedfurther.

Interestingly, we demonstrated that despite having both Krasand Pik3camutations at the time of tumor initiation from loss ofAPC, these cancers still develop through the adenoma-to-carci-noma sequence. Following inoculation of the colon with Adeno-Cre, a visible tumor can be identified in just a few weeks. Thesetumors are premalignant adenomas. The formation of the earlypolyp ismost likely related toWNT signaling (47). The loss ofAPCleads to the nuclear accumulation of CTNNB1, which results incellular proliferation and polyp formation. Without additionalgenetic events, the polyps tend to stabilize in size and do notprogress to cancers and may actually regress (48). With time, thevast majority of the tumors possessing mutations in KRAS,PIK3CA, or both will develop into invasive adenocarcinomas.The selective advantage conferred upon a cell carrying an addi-tional driver mutation has been estimated to increase the prob-ability of proliferation by 0.4% in silico (49). Because drivermutations are being identified in tumors containing less than109 cells (�1 cm in diameter), clones carrying thesemutations arelikely developing very early. This possibility might explain why astatistically significant change in the proliferation rate could notbe detected in the described experiments when additional drivermutations were added. Other factors including angiogenesis andstromal cell signalingmight also limit the proliferation rate in vivo.Interestingly, because driver mutations are identified in tumorscontaining less than 109 cells (�1 cm indiameter), clones carryingthese mutations are likely developing very early (50). For theseclones to outcompete others in the same tumor to a detectableextent, these additional driver mutations are likely occurringwhen the tumor consists of 104 cells or less. The small probabilityof proliferation causes dramatic changes as the number of cells inthese tumors enlarges. These driver mutations in vivo augmenttumor progression bymore than just increasing cell proliferation,but also by suppressing apoptosis, stimulating angiogenesis,recruiting stroma, and evading immune surveillance, amongothers (50).

The presence of KRAS and PIK3CA mutations at the time oftumor initiation in the setting of loss of APC does not eliminatethe formation of the premalignant intermediary. This is impor-tant because there is still the possibility to prevent invasion ofthese tumors if the polyps can be resected at colonoscopy. Inaddition, the malignant potential of these lesions might not beable to be predicted purely on tumor growth rate and mor-phology as it appears based on the experiments presented herethat loss of APC may be the key mediator of early tumorgrowth. The increased rate of tumor progression to invasivedisease in the setting of concomitant driver mutations indicatesthat it is likely beneficial to understand the biology of thepolyps that are removed from patients. Prognostic stratificationof premalignant lesions might be beneficial for determining thelikelihood of these tumors harboring foci of invasive diseaseand potentially the risk of future lesions developing. Furtherinvestigation is needed, but it might be necessary to screen

patients more frequently if they develop adenomas with high-risk molecular features.

The combinatorial accumulation of driver mutations,opposed to the timing of potential sequential mutations, hasthe greatest impact on tumor development and progression(49). The timing of when these mutations occur is very impor-tant, however, as this will impact the amount of time necessaryfor these lesions to become invasive cancers. In many instances,the canonical mechanism of tumorigenesis with mutationsslowly accumulating over time does likely occur. This studyand others demonstrate, though, that there are likely someinstances when the canonical pathway is not followed. Thiscould explain the development of interval cancers betweencolonoscopies. We have characterized an effective mouse mod-el for further investigations into the mechanisms of tumori-genesis that can ultimately be applied to the better under-standing of the molecular progression of colon tumors. Thesestudies will allow for improved risk stratification to dictatescreening intervals and the development of better chemo-preventive strategies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J.N. Hadac, A.A. Leystra, R.B. Halberg, D.A. DemingDevelopment of methodology: J.N. Hadac, A.A. Leystra, T.J. Paul Olson,R.B. Halberg, D.A. DemingAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.N. Hadac, A.A. Leystra, T.J. Paul Olson, A. Yueh,A.R. Schwartz, R.B. Halberg, D.A. DemingAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.N. Hadac, A.A. Leystra, M.E. Maher, A. Yueh,L. Clipson, K.A. Matkowskyj, R.B. Halberg, D.A. DemingWriting, review, and/or revision of the manuscript: J.N. Hadac, A.A. Leystra,T.J. Paul Olson, S.N. Payne, A. Yueh, L. Clipson, C.A. Pasch, K.A. Matkowskyj,R.B. Halberg, D.A. DemingAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M.E. Maher, S.N. Payne, D.M. Albrecht,L. Clipson, R.B. Halberg, D.A. DemingStudy supervision: R.B. Halberg, D.A. Deming

Grant SupportThis work was partially funded by Funk Out Cancer, a memorial event

dedicated to Kate Gates Falaschi. This work was also supported by theConquer Cancer Foundation of the American Society of Clinical Oncologythrough A Young Investigator Award (to D.A. Deming); T32 CA09017 (toT.J. Paul Olson), T32 CA009135 (to J.N. Hadac. and A.A. Leystra), R21CA170876 (to R.B. Halberg) and R01 CA123438 (to R.B. Halberg) fromthe NIH; 2012 AACR Career Development Award for Colorectal CancerResearch, Grant Number 12-20-01-HALB (to R.B. Halberg); start-up funds(to R.B. Halberg) from the UW Division of Gastroenterology and Hepa-tology, the UW Department of Medicine, and the UW School of Medicineand Public Health; and start-up funds (to D.A. Deming) from the UWCarbone Cancer Center, UW Department of Medicine, UW School ofMedicine and Public Health, and the UW Graduate School through theWisconsin Alumni Research Foundation; P30 CA014520 (Core Grant,University of Wisconsin Carbone Cancer Center); UW Carbone CancerCenter Gastrointestinal Disease Oriented Working Group (to R.B. Halbergand D.A. Deming); and Funk Out Cancer (to D.A. Deming).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 9, 2015; revised July 14, 2015; accepted July 15, 2015;published OnlineFirst August 14, 2015.

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References1. American Cancer Society. Cancer Facts & Figures 2014. Atlanta: American

Cancer Society; 2014.2. Nowell PC. The colonal evolution of tumor cell populations. Science

1976;194:23–8.3. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell

1990;61:759–67.4. Goss KH, Groden J. Biology of the adenomatous polyposis coli tumor

suppressor. J Clin Oncol 2000;18:1967–79.5. Kinzler KW, Vogelstein B. Cancer-susceptibility genes. Gatekeepers and

caretakers. Nature 1997;386:761, 763.6. Segditsas S, Tomlinson I. Colorectal cancer and genetic alterations in the

Wnt pathway. Oncogene 2006;25:7531–7.7. Markowitz SD, Bertagnolli MM. Molecular basis of colorectal cancer.

N Engl J Med 2009;361:2449–60.8. Parsons BL, Marchant-Miros KE, Delonqchamp RR, Verkler TL, Patterson

TA, McKinzie PB, et al. ACB-PCR quantification of K-RAS codon 12 GATand GTT mutation fraction in colon tumor and non-tumor tissue. CancerInvest 2010;28:364–75.

9. Smith G, Carey FA, Beattie J, Wilkie MJ V, Lightfoot TJ, Coxhead J,et al. Mutations in APC, Kirsten-ras, and p53—alternative geneticpathways to colorectal cancer. Proc Natl Acad Sci U S A 2002;99:9433–8.

10. Tang R, ChangchienCR,WuMC, FanCW, Liu KW,Chen JS, et al. Colorectalcancer without highmicrosatellite instability and chromosomal instability- an alternative genetic pathway to human colorectal cancer. Carcinogen-esis 2004;25:841–6.

11. Sweetser S, Smyrk TC, Sinicrope FA. Serrated colon polyps asprecursors o colorectal cancer. Clin Gastroenterol Hepatol 2013;11:760–7.

12. Imperiale TF, Ransohoff DF, Itzkowitz SH, Turnbill BA, Ross ME.Fecal DNA versus fecal occult blood for colorectal cancer screen-ing in an average-risk population. N Eng J Med 2004;351:2704–14.

13. Tomasetti C, Vogelstein B, Parmigiani G. Half or more of the somaticmutations in cancers of self-renewing tissues originate prior to tumorinitiation. Proc Natl Acad Sci U S A 2013;110:1999–2004

14. Moser AR, Pitot HC, Dove WF. A dominant mutation that predis-poses to multiple intestinal neoplasia in the mouse. Science1990;247:322–4.

15. Halberg RB, Katzung DS, Hoff PD, Moser AR, Cole CE, Lubet RA, et al.Tumorigenesis in the multiple intestinal neoplasia mouse: redundancy ofnegative regulators and specificity of modifiers. Proc Natl Acad Sci U S A2000;97:3461–6.

16. Halberg RB, Waggoner J, Rasmussen K, White A, Clipson L, PrunuskeAJ, et al. Long-lived Min mice develop advanced intestinal cancersthrough a genetically conservative pathway. Cancer Res 2009;69:5768–75.

17. Wong MH, Gordon JI. Genetic mosaic analysis based on Cre recombinaseand navigated laser capture microdissection. Proc Natl Acad Sci U S A2000;97:12601–6.

18. Jackson EL, Willis N, Mercer K, Bronson RT, Crowley D, MontoyaR, et al. Analysis of lung tumor initiation and progression usingconditional expression of oncogenic K-ras. Genes Dev 2001;15:3243–8.

19. Srinivasan L, Sasaki Y, Calado DP, Zhang B, Paik JH, DePinho RA, et al.PI3 kinase signals BCR-dependent mature B cell survival. Cell 2009;139:573–86.

20. Kuraguchi M, Wang XP, Bronson RT, Rothenberg R, Ohene-BaahNY, Lund JJ, et al. Adenomatous polyposis coli (Apc) is requiredfor normal development of skin and thymus. PLoS Genet 2006;2:1362–74.

21. MuzumdarMD, Tasic B,Miyamichi K, Li L, Luo L. A double-fluorescent Crereporter mouse. Genesis 2007;45:593–605.

22. BeckerC, FantiniMC,Wirtz S,NikolaevA, KiesslichR, LehrHA, et al. In vivoimaging of colitis and colon cancer development in mice using highresolution chromoendoscopy. Gut 2005;54:950–4.

23. Hung KE, MaricevichMA, Richard LG, ChenWY, RichardsonMP, Kunin A,et al. Development of a mouse model for sporadic and metastatic colontumors and its use in assessing drug treatment. Proc Natl Acad Sci U S A2010;107:1565–70.

24. Pickhardt PJ, Halberg RB, Taylor AJ, Durkee BY, Fine J, Lee FT, et al.Microcomputed tomography colonography for polyp detection in anin vivo mouse tumor model. Proc Natl Acad Sci U S A 2005;102:3419–22.

25. Paul Olson TJ, Hadac JN, Sievers CK, Leystra AA, Deming DA, Zahm CD,et al. Dynamic tumor growth patterns in a novel murine model ofcolorectal cancer. Cancer Prev Res 2014;7:105–13.

26. The Cancer Genome Atlas Network. Comprehensive molecular char-acterization of human colon and rectal cancer. Nature 2012;487:330–7.

27. Velho S,MoutinhoC, Cirnes L, AlbuquerqueC,Hamelin R, Schmitt F, et al.BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps andcancer: primary or secondary genetic events in colorectal carcinogenesis?BMC Cancer 2008;8:255.

28. Leystra AA, Deming DA, ZahmCD, FarhoudM, Olson TJP, Hadac JN, et al.Mice expressing activated PI3K rapidly develop advanced colon cancer.Cancer Res 2012;72:2931–6.

29. Haigis KM, Kendall KR, Wang Y, Cheung A, Haigis MC, Glickman JN,et al. Differential effects of oncogenic K-Ras and N-Ras on proliferationdifferentiation and tumor progression in the colon. Nat Genet 2008;40:600–8.

30. Janssen KP, Alberici P, Fsihi H, Gaspar C, Breukel C, Franken P, et al. APCand oncogenic KRAS are synergistic in enhancing Wnt signaling in intes-tinal tumor formation and progression. Gastroenterology 2006;131:1096–109.

31. Deming DA, Leystra AA, Nettekoven L, Sievers C, Miller D, MiddlebrooksM, et al. PIK3CA and APCmutations are synergistic in the development ofintestinal cancers. Oncogene 2014;33:2245–54.

32. Abramovitch R, Marikovsky M, Meir G, Neeman M. Stimulation oftumour growth by wound-derived growth factors. Br J Cancer 1999;79:1392–8.

33. Schek RM, Hollister SJ, Krebsbach PH. Delivery and protection of adeno-viruses using biocompatible hydrogels for localized gene therapy.Mol Ther2004;9:130–8.

34. Hu WW, Wang Z, Hollister SJ, Krebsbach PH. Localized viral vectordelivery to enhance in situ regenerative gene therapy. Gene Ther 2007;14:891–901.

35. Siemens DR, Elzey BD, Lubaroff DM, Bohlken C, Jensen RJ, Swanson AK,et al. Cutting edge: restoration of the ability to generate CTL in miceimmune to adenovirus by delivery of virus in a collagen-based matrix.J Immunol 2001;166:731–5.

36. Dai Q, Manfield L, Wang Y, Murrell GAC. Adenovirus-mediated genetransfer to healing tendon—enhanced efficiency using a gelatin sponge.J Orthop Res 2003;21:604–9.

37. Rubin EM, Raptopoulos VD. Images in clinical medicine. The virtual applecore of a colonic carcinoma. N Engl J Med 2005;352:2733.

38. Powell SM, Zilz N, Beazer-Barclay Y, Bryan TM, Hamilton SR, ThibodeauSN, et al. APC mutations occur early during colorectal tumorigenesis.Nature 1992;359:235–7.

39. Samowitz WS, Slattery ML, Sweeney C, Herrick J, Wolff RK, Albertsen H.APC mutations and other genetic and epigenetic changes in colon cancer.Mol Cancer Res 2007;5:165–70.

40. Harada N, Tamai Y, Ishikawa T, Sauer B, Takaku K, Oshima M, et al.Intestinal polyposis in mice with a dominant stable mutation of theb-catenin gene. EMBO J 1999;18:5931–42.

41. Loeve F, Boer R, Zauber AG, Van Ballegooijen M, Van Oortmarssen GJ,Winawer SJ, et al. National Polyp Study data: evidence for regression ofadenomas. Int J Cancer 2004;111:633–9.

42. Pickhardt PJ, Kim DH, Pooler BD, Hinshaw JL, Barlow D, Jensen D, et al.Assessment of volumetric growth rates of small colorectal polyps with CTcolonography: a longitudinal study of natural history. Lancet Oncol2013;14:711–20.

43. Pritchard CC, Grady WM. Colorectal cancer molecular biology moves intoclinical practice. Gut 2011;60:116–29.

44. Jones S, Chen W-D, Parmigiani G, Diehl F, Beerenwinkel N, Antal T, et al.Comparative lesion sequencing provides insights into tumor evolution.Proc Natl Acad Sci U S A 2008;105:4283–8.

45. Wood LD, Parsons DW, Jones S, Lin J, Sj€oblom T, Leary RJ, et al. Thegenomic landscapes of human breast and colorectal cancers. Science2007;318:1108–13.

Hadac et al.

Cancer Prev Res; 8(10) October 2015 Cancer Prevention Research960

46. Yamada S, Yashiro M, Maeda K, Nishiguchi Y, Hirakawa K. A novelhigh-specificity approach for colorectal neoplasia: detection of K-ras2oncogene mutation in normal mucosa. Int J Cancer 2005;113:1015–21.

47. SmithKJ, JohnsonKA,Bryan TM,HillDE,Markowitz S,Wilson JK, et al. TheAPC gene product in normal and tumor cells. Proc Natl Acad Sci U S A1993;90:2846–50.

48. Bozic I, Antal T, OhtsukiH, Carter H, KimD, Chen S, et al. Accumulation ofdriver and passenger mutations during tumor progression. Proc Natl AcadSci U S A 2010;107:18545–48.

49. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70.

50. Vogelstein B, Papdopoulos N, Velculescu VE, Zhou S, Diaz LA, Kinzler KW.Cancer genome landscapes. Science 2013;339:1546–58.

www.aacrjournals.org Cancer Prev Res; 8(10) October 2015 961

Timing of Driver Mutations versus Tumor Progression