The use of genetic programming in the analysis of quantitative gene expression profiles for...

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Open AcceResearch articleThe use of genetic programming in the analysis of quantitative gene expression profiles for identification of nodal status in bladder cancerAnirban P Mitra†1, Arpit A Almal†2, Ben George3, David W Fry2, Peter F Lenehan2, Vincenzo Pagliarulo4, Richard J Cote1, Ram H Datar*1 and William P Worzel2
Address: 1Department of Pathology, University of Southern California Keck School of Medicine, 2011 Zonal Avenue, HMR 312, Los Angeles CA 90033, USA, 2Genetics Squared Inc., 210 South 5th Avenue, Suite A, Ann Arbor MI 48104, USA, 3Department of Internal Medicine, Gundersen Lutheran Medical Center, 1900 South Avenue, La Crosse WI 54601, USA and 4Dipartimento Emergenza e Trapianti d'Organo, Sezione di Urologia, Università di Bari, Piazza G. Cesare 11, Bari 70124, Italy

Email: Anirban P Mitra - [email protected]; Arpit A Almal - [email protected]; Ben George - [email protected]; David W Fry - [email protected]; Peter F Lenehan - [email protected]; Vincenzo Pagliarulo - [email protected]; Richard J Cote - [email protected]; Ram H Datar* - [email protected]; William P Worzel - [email protected]

* Corresponding author †Equal contributors

AbstractBackground: Previous studies on bladder cancer have shown nodal involvement to be an independent indicator ofprognosis and survival. This study aimed at developing an objective method for detection of nodal metastasis frommolecular profiles of primary urothelial carcinoma tissues.

Methods: The study included primary bladder tumor tissues from 60 patients across different stages and 5 controltissues of normal urothelium. The entire cohort was divided into training and validation sets comprised of node positiveand node negative subjects. Quantitative expression profiling was performed for a panel of 70 genes using standardizedcompetitive RT-PCR and the expression values of the training set samples were run through an iterative machine learningprocess called genetic programming that employed an N-fold cross validation technique to generate classifier rules oflimited complexity. These were then used in a voting algorithm to classify the validation set samples into those associatedwith or without nodal metastasis.

Results: The generated classifier rules using 70 genes demonstrated 81% accuracy on the validation set when comparedto the pathological nodal status. The rules showed a strong predilection for ICAM1, MAP2K6 and KDR resulting in geneexpression motifs that cumulatively suggested a pattern ICAM1>MAP2K6>KDR for node positive cases. Additionally, themotifs showed CDK8 to be lower relative to ICAM1, and ANXA5 to be relatively high by itself in node positive tumors.Rules generated using only ICAM1, MAP2K6 and KDR were comparably robust, with a single representative rule producingan accuracy of 90% when used by itself on the validation set, suggesting a crucial role for these genes in nodal metastasis.

Conclusion: Our study demonstrates the use of standardized quantitative gene expression values from primary bladdertumor tissues as inputs in a genetic programming system to generate classifier rules for determining the nodal status.Our method also suggests the involvement of ICAM1, MAP2K6, KDR, CDK8 and ANXA5 in unique mathematicalcombinations in the progression towards nodal positivity. Further studies are needed to identify more class-specificsignatures and confirm the role of these genes in the evolution of nodal metastasis in bladder cancer.

Published: 16 June 2006

BMC Cancer 2006, 6:159 doi:10.1186/1471-2407-6-159

Received: 09 February 2006Accepted: 16 June 2006

This article is available from: http://www.biomedcentral.com/1471-2407/6/159

© 2006 Mitra et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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BackgroundCancer of the urinary bladder is the seventh most com-mon cancer worldwide (3.2% of all cancers), with an esti-mated annual incidence of 330,000 new cases and towhich 179,000 deaths are attributed each year [1,2]. In theUSA, where more than 63,000 new cases of bladder cancerwere expected in 2005, urothelial carcinoma (UC) is themost common histology (90%), followed by squamouscell carcinoma (6–8%), adenocarcinoma (2%), and avariety of other rare tumor types [3]. The standard TNMclinical stage classification system for bladder cancer rec-ommended by the American Joint Committee on Cancertakes into account the depth of invasion of the bladderwall by the primary tumor (T), the presence and size ofmetastatic regional lymph nodes (N), and the presence orabsence of distant metastases (M) [4]. Nodal involvementis considered to be an independent risk factor for recur-rence and survival after cystectomy for organ-confinedbladder cancer [5]. Consequently, extensive bilateral pel-vic lymphadenectomy is now considered an integral partof the surgery, having been shown to significantlyimprove the prognosis of patients with muscularis pro-pria-invasive bladder cancer [6,7]. Non-muscularis pro-pria-invasive tumors (TNM Stages 0a, 0is, and I), confinedto the bladder mucosa or subepithelial connective tissue(pTa, pTis, and pT1) without regional (N0) or distant(M0) metastases, are generally treated by transurethralresection of the tumor with fulguration, intravesicalchemotherapy, and radiotherapy. Although cures are pos-sible, up to 80% of these presumed "localized" tumorswill eventually recur following initial resection, with up to25% progressing to muscularis propria-invasive disease[8]. The confirmation of the existing true nodal status in apatient with bladder cancer thus assumes primary impor-tance, along with the need to determine if the tumor hasthe molecular potential to metastasize to the lymph nodeslater, provided undiagnosed micrometastasis has notoccurred already.

Molecular changes in bladder cancer have been shown toprecede morphologic changes that can be identified visu-ally [9]. Further, some tumors have specific molecular pat-terns that predispose them to be more morphologicallyaggressive, with a greater propensity to metastasize andrecur, regardless of their clinical stage at diagnosis [10].Hence, morphologic changes need to be complementedwith molecular correlates for an accurate prediction ofbladder tumor progression.

The goal of this study was to create an objective and accu-rate tool for the identification of nodal status from pri-mary tumor tissue. Since bladder cancer has a multi-factorial etiology with a complex pathogenesis encom-passing various pathways that involve more than a simpletwo directional (up/down) regulation of a few genes, we

felt that it was necessary to investigate a comprehensivepanel of genes to define this complex disease. Utilizingbladder tissue biopsies from 60 primary UC subjects andfive normal controls, our study involved the analysis of aset of 70 candidate markers involved in crucial pathwaysthat have been shown to be deregulated in cancer, includ-ing those of cell cycle regulation, apoptosis, angiogenesis,invasion and metastasis, and anti-oxidation [Figure 1,Additional file 1] [11,12]. Since scaling the gene expres-sion levels to represent fold changes relative to a basevalue could have biased the significance of these genechanges, there was a concern that representing the data inthis way might obscure any correlation with the alteredgene's function. We, therefore, adopted a standardizedcompetitive reverse transcriptase – polymerase chain reac-tion (StaRT-PCR™) approach to quantitatively measuregene expression values in relation to a million moleculesof a housekeeping gene like β-actin [11]. This gave us anexpression profile and molecular signature for each tissuesample with the lowest inter-sample and intra-sample var-iability.

Using a machine learning technique called genetic pro-gramming (GP) [13], the gene expression values werethen used to classify the primary tumor tissue samplesinto those associated with nodal involvement (node pos-itive, NP) and those from subjects known to have nonodal involvement (node negative, NN). GP uses theavailable data to produce a set of classifiers ("rules") thatare optimized in an iterative fashion through successiveretention of the better performing rules. One of the keycharacteristics of GP is its ability to automatically selectvariables and operators and assemble them into appropri-ate structures that form predictive functions for classifyingthe samples, often discovering unusual and unexpectedcombinations of input variables. In this study, the sam-ples were divided into training and validation sets, and GPwas used in a supervised learning mode on the training setto develop a discriminant classifier solution which thenused the validation set to test the generality of the solutionproduced. We, herein, report that by employing GP toanalyze quantitative gene expression profiles of primarytumor tissue, one can accurately determine the nodal sta-tus of bladder cancer in the same patient, thereby enhanc-ing the ability to correctly assess the extent of disease.

MethodsPatient population and distributionThe study cohort was comprised of 60 UC subjects andfive normal controls (n = 65). UC tissue was obtainedfrom 50 subjects who underwent radical cystectomy forUC of the bladder at the University of Southern Califor-nia/Norris Comprehensive Cancer Center from 1997 to2001 and from 10 subjects who underwent treatment forpTa and pT1 bladder cancer at the University of Califor-



nia, San Francisco. Nodal staging was determined by his-topathological examination in the former group and byimaging in the latter. Controls consisted of normalurothelium from the bladder neck of five subjects whounderwent radical prostatectomy for prostatic adenocarci-noma localized to the prostate with no bladder involve-ment at the Norris Comprehensive Cancer Center. Noneof these subjects had any history of bladder cancer. The 60tumors had the following stage distribution: pTa (n = 10),pT1 (n = 13), pT2 (n = 8), pT3 (n = 22) and pT4 (n = 7);21 of these 60 subjects (35%) were NP, although none of

the subjects had distant metastatic disease. Subjects withpure adenocarcinoma, squamous cell carcinoma, or smallcell carcinoma were not included in the analysis. Nodalstatus of the subjects was determined during initial diag-nosis and after pelvic lymphadenectomy during radicalcystectomy for the invasive UC cases. Pathological stagewas determined according to the tumor-node-metastasis(TNM) system [4]. Primary tumor samples from the cys-tectomy specimens were preserved as archival paraffin-embedded tissue blocks and were available in all cases.Informed consent was obtained from all subjects. The

Marker panel employed for standardized competitive RT-PCR analysisFigure 1Marker panel employed for standardized competitive RT-PCR analysis. A total of 70 genes involved in eight broad pathways commonly deregulated in cancer were chosen for this study. The primary effector pathways of tumorigenesis encom-pass apoptosis, cell cycle, gene regulation, cell growth regulation and anti-oxidation, and are comprised of 57 genes. There is a significant overlap of markers among the first three pathways. The secondary effector pathways include signal transduction, angiogenesis and invasion, and are comprised of 13 genes. All the listed genes exert stimulatory, inhibitory and/or regulatory effects on their respective pathway(s).

ANXA5BAD

BCL2BCL2L1

CYP1A2DAP

PTGS2TGFBR2TGIFTNF

TNFAIP1TNFRSF1A

TNFSF10TRAF4

CCNA2CCND3

CCNE1CCNG1

CDC2CDC25C

CDK7CDK8

PCNA

RB1 RBL2CDKN1A CDKN2ACDKN1B CDKN2CGAPDH MXD1

TP53

E2F1E2F2 E2F4

E2F5

FOS JUNFOSL1 JUNBHSF1 MAX

MAP3K14 MYCSP1 NFKB1

ERBB2MAPK8MAPK9

MAPK12MAP2K6STAT3

LYN

IGF1IGF2RPDGFB

PDGFRL

GSTM3GSTP1GSTT1SOD1

FGF5FGFR4VEGFKDR

BMP6CDH3ICAM1MMP16TIMP2

NORMALTISSUE

LOCO – REGIONALTUMOR GROWTH

DISTANTMETASTASIS

Apoptosis Gene Regulation

Cell Cycle Signal Transduction

Apoptosis + Cell Cycle

Apoptosis + Cell Cycle + Gene Regulation

Anti-oxidation Angiogenesis

Cell Growth Regulation Invasion

Color codes of pathways involved in tumorigenesis

PRIMARY EFFECTOR PATHWAYS

SECONDARY EFFECTOR PATHWAYS



gene expression profiling studies were approved by theUniversity of Southern California and the University ofCalifornia, San Francisco Institutional Review Boards.

The total study population was divided into training andvalidation sets. The former was comprised of 11 NP sub-jects and 23 NN subjects, while the latter consisted of 10NP subjects and 21 NN subjects. The 44 NN subjectsincluded the 5 normal controls which were classified asNN for the purpose of analysis in this study. The distribu-tion of the subjects across both sets and nodal classes wasmade to maintain an approximately equal proportionacross all tumor staging strata [Table 1].

RNA extraction and cDNA synthesisRNA was extracted using the TRIzol® method (Invitrogen,Carlsbad, CA, USA). Formalin-fixed paraffin embeddedtissue sections were lysed with a syringe in TRIzol®. 400 μLof chloroform was then added followed by centrifugationto separate the RNA containing aqueous phase. This wasfollowed by addition of linear acrylamide (Ambion, Aus-tin, TX, USA) that served as a carrier and 1 mL of isopro-panol to precipitate the RNA followed by incubation at -80°C for two hours. The tubes were then thawed and cen-trifuged at 4°C and the supernatant was removed. TheRNA pellet was washed with cold 70% ethanol followedby centrifugation and removal of the supernatant. TheRNA pellet was dried and resuspended in DEPC-treatedwater. DNase treatment was then performed using DNA-free™ (Ambion, Austin, TX, USA) following the manufac-turer's instructions. cDNA was prepared as described pre-viously [14].

StaRT-PCR™, image analysis and quantitationQuantitative gene expression profiling was done usingStaRT-PCR™ analysis as described previously [14]. Theinternal standard competitive template (CT) mixtures (A-F) over six logs of concentration were obtained from Gene

Express, Inc. (Toledo, OH, USA). While each of the sixmixtures (A-F) contained internal standard CTs for 381target genes in addition to 600,000 β-actin CT molecules/μL, our study targeted a list of 70 transcripts [Figure 1,Additional file 1]. For each sample, StaRT-PCR™ analysiswas performed using five different CT mixes (B-F). Thuseach sample underwent five separate PCR analyses; eachseparate reaction containing the ready-to-use master mix-ture, cDNA sufficient for expression measurements of the71 transcripts (including β-actin), primers for the 71 tran-scripts and one of the five CT mixes (B-F). Following PCR,the amplification products were electrophoresed, andimage analysis and quantitation of band fluorescenceintensities were done as described previously [14].

Genetic program analysis, voting algorithm and gene usage frequencyGP was used in a supervised learning mode on the train-ing set to develop classifier programs. For this, a "geneticpool" of candidate classification programs was createdfrom which future programs were created through selec-tion and re-combination. The programs were initially cre-ated by randomly choosing inputs and arithmetic andBoolean operators that work with the type of inputsselected. A small subgroup of programs was then selectedfrom the main population to create a "mating pool" ofprograms. Each program was evaluated on input data andthe output was a prediction of the nodal status associatedwith these inputs. The accuracy of a program in correctlyclassifying the samples according to pre-specified labelswas used to calculate a fitness measure for the program.Fitness was determined by calculating the area under thecurve (AUC) for the receiver operating characteristic(ROC) of a program generated by the GP system, and evo-lution was driven to maximize the AUC so as to yield ruleswith high sensitivity and specificity. The complexity of therules generated was also restricted to prevent overfitting.This was done by the strict use of mathematical operators

Table 1: Distribution of the study population on the basis of nodal positivity and tumor stage.

Tumor Stage Training Set Validation Set

Node positive Node negative Total Node positive Node negative Total

Normal controls 3 3 2 2pTa 0 3 3 0 7 7pT1 2 6 8 1 4 5pT2 0 4 4 0 4 4pT3 7 5 12 7 3 10pT4 2 2 4 2 1 3

Grand total 11 23 34 10 21 31

The total cohort of 65 subjects included five normal controls that were classified as node negative. An approximately equal distribution of the subjects was attempted between both sets in all tumor stages and nodal classes to eliminate bias. Tumor and nodal stages was determined according to the American Joint Committee on Cancer recommended TNM system for urinary bladder cancer (2002).



(e.g., +, -, x, /, exp), logical operators (e.g., 'and', 'not', 'or')and comparison operators (e.g., =, >, <, ≥, ≤), and geneusage was restricted to no more than seven genes in a solu-tion. 'exp' is an exponent function where exp(N) is equiv-alent to eN. While this may seem odd, it expresses theexponential quality of response relationships betweengenes, particularly within a pathway. The '?' operator wasused as a conditional phrase "IF <predicate> THEN<expression1> ELSE <expression2>."

The two programs in the mating pool with the highest fit-ness values were then chosen for selective combination(i.e., mating) to produce offspring. The offspring pro-grams then replaced the least fit programs in the mainpopulation, potentially containing superior traits takenfrom each of their parents. This was repeated and, overmany generations, progressively better programs were cre-ated [Figure 2]. The relatively small data set also necessi-tated the employment of a cross validation technique toestimate the ability of the classifier to generalize to unseensamples, giving an approximation of its robustness. Thiswas done using the N-Fold cross validation techniquewherein the training set was subdivided into eleven"folds" (N = 11), as there were eleven NP cases in thetraining set. Classifier rules were developed using the sam-ples in 10 folds and each rule was then tested on the elev-

enth fold of the training set. The process was repeated 10more times with each fold taking a turn as the test fold. 20runs of 11 folds each were completed and the set of clas-sifiers that had the best total performance across all thefolds was then selected. As GP is a stochastic process andgives more than one solution with the same accuracy, thisgave a reasonable sample of the best performing classifiersets. Classifier sets ("meta-rules") were then used in amajority voting scheme to classify the samples in the val-idation set. Aggregate performance of these meta-rules onthe test folds was taken as the predictor of the classifica-tion error, and the selected meta-rule was the one with theleast test error. The 11-fold cross validation resulted in ameta-rule for each run that was composed of eleven rules,one for each fold. The meta-rule then "voted" for a samplepresented to it. If the majority of the rules (i.e., six ormore) voted that the sample belonged to the target class(in this case, NP), the meta-rule was designated as predic-tive of the target class.

Running the expression values of the training set samples20 times over 11 folds resulted in 220 rules, each of whichhad five genes on an average. Thus, the postulated fre-quency of occurrence of each of the 70 genes in 220 rulesif all had equal probabilities of being selected was 15.71.The actual frequencies of occurrence of all genes wererecorded by identifying the number of instances amongstall the 220 rules, wherein a rule had the gene as one of itsconstituents. Since the occurrence of each gene in a rulewas a binary event, the frequency of each gene beingselected followed a binomial distribution enabling thecalculation of binomial probabilities and their corre-sponding p-values. Statistical calculations were performedusing SAS (release 9.1).

ResultsGeneration of rulesQuantitative gene expression profiling of the tissue sam-ples for the above mentioned 70 genes was done usingStaRT-PCR™ and the expression values for the samplesgrouped under the training set were run 20 times throughthe GP system for 100 generations over 11 folds to yieldmeta-rules. The fitness measure tried to maximize theAUC while overfitting was avoided by using simple math-ematical functions and restricting rule sizes as describedabove. N-fold cross validation was used as the resamplingtechnique to test the overall generality of the classifier.The generated rules were then subject to a majority votingalgorithm and the best performing rules were chosen andtested on the validation set against the histopathologicallydetermined nodal status. The accuracy of each meta-rulewas assessed by calculating how well it classified the vali-dation set samples based solely on their molecular charac-teristics. The final meta-rule thus generated is shown inTable 2 which correctly identified 6 out of 10 NP samples

The genetic programming processFigure 2The genetic programming process. This iterative tech-nique was employed on the training set samples to generate classifier rules that were tested on the validation set. Ran-domly chosen components were initially used to create a population of candidate programs from which a small mating pool of candidate programs was generated. Inputs were passed into these programs and the predicted nodal statuses were evaluated for fitness. The two best performing pro-grams were then mated to produce offspring that replaced the two least fit programs. This process was repeated over many generations to create better programs.

Population of candidate programs

Run programs in group

Molecular characteristics

Output classMate parents to create offspring

Results compared to produce fitness

Replace two least fit programs with offspring

Inputs passed to programs

Select two best programs as parents

Select mating group

Create population of programs



and 19 out of 21 NN samples in the validation set, result-ing in a positive predictive value of 75% and negative pre-dictive value of 83% [Table 3]. All of the normal cases,classified as NN, were correctly identified as being nodenegative, yielding an overall sensitivity of 60% and a spe-cificity of 90%.

Gene usageCross validation consistency is a concept that implies thatthe presence of a fundamental phenomenon implicit inthe data will be reflected by a similarity between theresults for different folds [15]. The frequency of geneusage in the best rules across many runs with differentfold compositions of the data thus allowed us the possi-bility of identifying the most important genes as well asgene-gene interactions used in the classifiers for target dis-crimination. Although the stochastic nature of GP com-bined with the fact that the fold composition is partlydetermined by random selection may lead one to assumethat it might be possible to craft a rule that could classifyany limited number of samples given a random selectionof genes, this proves not to be the case. We show thatrepeatedly running the system and tabulating the use ofgenes across all folds in all runs reveals a strong preferencefor certain genes. The genes KDR, MAP2K6 and ICAM1(encoding the kinase insert domain receptor, mitogen-

activated protein kinase kinase 6, and intercellular adhe-sion molecule 1, respectively) showed a strong predilec-tion to be used among the 220 rules drawn from 20 runsof 11 folds [Figure 3]. Since the presence or absence of agene in a rule is a binary event, the frequency of each genebeing selected follows a binomial distribution. The bino-mial probabilities of the above genes were 9.69E-130,1.13E-110, and 4.10E-78, respectively, with one-sided p-values of <0.00001 against the null hypothesis of each ofthe 70 genes having an equal probability of being selected[Table 4]. The p-values for the next five genes were alsolow enough to indicate significant selectivity towardsthem. Examination of the rules presented in Table 2 showthe prevalence of the top three genes where each rule onthis list uses at least one of these genes and four of therules use all three genes, though in different combina-tions.

Gene expression motifsIn addition to raw gene frequency information, the rulesdeveloped were examined for recurring mathematicalcombinations that may be called "motifs". These not onlyshowed the tendency to utilize certain genes more thanothers but also demonstrated the relationship betweenthese genes. From this, it may be possible to identify path-ways that are associated with the signature of a certain tar-

Table 3: Performance of the selected meta-rule generated from the set of 70 genes on the validation set and result metrics.

Pathologically Node Positive Pathologically Node Negative

Predicted Node Positive by GP using 70 genes 6§ 2Predicted Node Negative by GP using 70 genes 4 19†

§ True positive subjects.† True negative subjects.Accuracy: 81%Sensitivity: 60%Specificity: 90%Positive Predictive Value: 75%Negative Predictive Value: 83%

Table 2: Final meta-rule for node positive patients generated from the set of 70 genes.

Rule number Classifier Rule

1 exp(exp(HSF1)) - exp(MXD1)/(KDR - MAP2K6) > 2.7182 (MAP2K6/KDR) × (exp(TGIF) - MAP2K6/ICAM1) > .7093 (ICAM1 - CDK8)/(exp(JUNB) × (JUNB - exp(TGFBR2))) > 1.324 ANXA5 × MAP2K6/(KDR × (ICAM1 - CDK8)) > 1.7015 (ICAM1 - MAP2K6) × exp(MAP2K6 - KDR) > 3653.8136 (ICAM1 - CDK8) × TP53/(exp(TGFBR2) × PTGS2) > 21941.4537 (CCND3/MAP2K6) × (exp(BMP6) - (KDR/MAP2K6)) > .2018 MAP2K6/(CDKN1A × exp(MAPK12) × (CDC25C - KDR)) > 7.7039 (ANXA5 - exp(PDGFRL))/(CDKN1A × (KDR - exp(TGFBR2))) > .04410 ANXA5/(CDKN1A × (exp(PTGS2) - (CDK8/ICAM1))) > 79.00211 MAP2K6/(KDR × (ICAM1 - (TNFAIP1/exp(PDGFB)))) > 1.182



get class, which in this study was the class of node positivesubjects.

Examination of the rules revealed a consistent relation-ship between the expression levels of the three most fre-quently utilized genes, i.e., KDR, MAP2K6 and ICAM1.That is, the more highly expressed MAP2K6 and ICAM1were when compared to KDR, the more likely it was thata sample would be NP. These, however, are relative com-parisons. In other words, KDR is lower when compared toMAP2K6 and ICAM1, but a simple assumption of lowKDR expression in general cannot be construed as amarker of nodal positivity (see Additional file 2). Anexample motif that shows this relationship is the expres-sion 'MAP2K6/KDR'. The rules developed all take theform: "IF [mathematical expression] ≥ [threshold] THENNP". In this case, a lower KDR expression value translatesto a higher ratio with an increasing likelihood of the sam-ple being classified as NP. Similarly, the higher MAP2K6is when compared to KDR, the more likely it is that thisexpression will be greater than the constant value. Thismotif occurs in rules 2, 4, 7 and 11 in Table 2. A similarmotif that was often observed was 'MAP2K6 - KDR', as inrules 5 and 1 [Table 2]. (Note that in Rule 1, the relation-

ship is reversed because it appears in the denominator ofthe ratio.) Again, KDR would tend to be mathematicallylower than MAP2K6.

The motif 'MAP2K6/ICAM1' and its related motif 'ICAM1- MAP2K6' appear quite frequently, with the former usedin a reductive way (i.e., reducing the value that is com-pared to the constant value) and the latter in an additiveway (i.e., increasing the value that is compared to the con-stant value). This suggests that in NP cases,ICAM1>MAP2K6. Since we had previously identified therelationship MAP2K6>KDR from the use of the MAP2K6and KDR motifs described above, we can infer that in NPcases ICAM1>MAP2K6>KDR. This might be called "genetransitivity" in the sense that since MAP2K6 is greater thanKDR but less than ICAM1, we can infer that ICAM1 is ingeneral greater than KDR in NP cases. This relationshipmay be seen in rules 2 and 5, respectively [Table 2].

Another motif observed was 'ICAM1 - CDK8' or the simi-lar motif 'ICAM1/CDK8', which were featured in rules 3, 4and 6 [Table 2]. The prevalence of these motifs shows thata high ICAM1 value relative to CDK8 (which encodes cyc-lin-dependent kinase 8) is likely in NP samples. Rule 10shows the motif 'CDK8/ICAM1', but as this expression isbeing subtracted from the rest of the expression, it alsoleads to the same conclusion of ICAM1 levels tending tobe higher relative to CDK8. There was no obvious motifthat linked CDK8's relationship to either MAP2K6 orKDR.

Finally, though not technically a motif, the fourth mostfrequently used gene, ANXA5, that encodes for annexinA5, appeared in a variety of combinations in rules 4, 9 and10 [Table 2] and a relatively high expression value of thegene was generally associated with NP cases.

Identification of a single ruleWhile the voting algorithm seems to work reliably, thereis a natural desire to identify the "best" rule for classifyingsamples. While this may be contrary to the population-based process used by GP, the gene usage frequencyresults indicate that the top three genes are used signifi-cantly more often than the others. This suggests that thesegenes play the most important part in distinguishing NPsamples from NN samples. One hypothesis could be thatthese genes are carrying the bulk of the value in the rulespresented. To investigate this idea further, rules were cre-ated using only these three genes viz., KDR, MAP2K6 andICAM1, as inputs. The entire GP process was repeated inorder to clearly identify the relationship of these threegenes and to test the robustness of the rules developedfrom this subset of genes. The final meta-rule obtainedafter 11-fold cross validation comprised of eleven rules(see Additional file 3).

Histogram of Gene Usage FrequenciesFigure 3Histogram of Gene Usage Frequencies. Examination of the gene usage frequencies among the best of 220 rules drawn from 20 runs of 11 folds showed a strong preference for the KDR, MAP2K6 and ICAM1 genes which were also components of some of the major gene expression motifs. Rules created using only the top three genes showed a com-paratively better performance, indicating their importance in the genesis of nodal metastasis.



The performance of these rules on the validation set wasfound to be comparable to the previous results generatedusing the panel of 70 genes, demonstrating equal levels ofaccuracy and comparable sensitivity and specificity met-rics [Table 5], which tend to support the hypothesis thatrules created with a reduced number of genes that arebelieved to be the key modulators of the target processgenerate equally robust classifiers. Of note is the fact thatthe first rule from the generated meta-rule (see Additionalfile 3) when singularly analyzed retrospectively on the val-idation set, produced a markedly better result with 70%sensitivity and 100% specificity. The positive predictivevalue of this rule was 100% and the negative predictivevalue was 88%, though there was nothing that uniquely

stood out about this rule in comparison with the others(see Additional file 4). However, the observation thatmost of the rules show similarity in constitution withrespect to gene usage further bolsters the hypothesis thatthese genes are critical in the development of nodal metas-tasis and interact with each other in distinct effector path-ways.

DiscussionRecent studies suggest that the significant relapse rates forbladder tumors that do or do not invade the muscularispropria may be related to the presence of micrometastasesin pelvic lymph nodes that are undetectable using conven-tional computed tomography, magnetic resonance imag-

Table 5: Performance of the meta-rule generated using the three most frequently used genes, viz. KDR, MAP2K6 and ICAM1, on the validation set and result metrics.

Pathologically Node Positive Pathologically Node Negative

Predicted Node Positive by GP using 3 genes 7§ 3Predicted Node Negative by GP using 3 genes 3 18†

§ True positive subjects† True negative subjectsAccuracy: 81%Sensitivity: 70%Specificity:86%Positive Predictive Value: 70%Negative Predictive Value: 86%

Table 4: Probability of gene usage from the set of 70 genes due to random chance.

Gene Actual occurrence of gene (in 220 rules) Binomial probability p-value (one-sided)

KDR 159 9.69E-130 <0.00001MAP2K6 146 1.13E-110 <0.00001ICAM1 121 4.10E-78 <0.00001ANXA5 60 7.04E-20 <0.00001CDK8 56 3.38E-17 <0.00001

CDKN1A 55 1.49E-16 <0.00001JUNB 49 6.56E-13 <0.00001

TGFBR2 41 1.08E-08 <0.00001TNF 24 1.11E-02 0.01490

TNFAIP1 23 1.76E-02 0.02599CCND3 19 6.73E-02 0.16020PDGFRL 18 8.23E-02 0.22749MAPK12 15 1.04E-01 0.49264GAPDH 15 1.04E-01 0.49264PTGS2 12 7.05E-02 0.20295PDGFB 11 5.26E-02 0.13246

Number of genes per rule = 5 (approximately)

Arranged in decreasing order of their frequencies of occurrence in 220 rules, the genes show a general trend towards increasing probability of being selected in a rule by random chance. The postulated frequency of occurrence of each gene if all have equal probabilities of being selected is 15.71. PTGS2 and PDGFB have smaller probabilities than the two genes preceding them because they were actually used less frequently than random chance would suggest.

Random occurrence of gene Number of rulesNumber of genes = × pper rule

Total number of genes

= × =2205

7015 71.



ing, positron emission tomography and routinehistopathologic examination [16,17]. Hence, considera-tion for early cystectomy with pelvic lymphadenectomy isnow being advocated even for "localized" bladder cancersthat have not invaded the muscularis propria [18]. A moreaccurate definition of the nodal status upon initial diag-nosis and during follow-up of bladder cancer will go along way in minimizing the significant understaging andoverstaging that appears to currently exist and thereby bet-ter equipping the clinician with the tools needed to deter-mine the optimal treatment and follow-up strategies for aparticular patient.

Over the past decade, efforts have begun to identifymolecular markers that can predict the propensity of blad-der tumors to metastasize to the lymph nodes. While sin-gle molecular markers with significant correlations havebeen identified, the predictive and prognostic potentialoffered by them is still not optimal. The current situationwarrants the need to generate a panel of markers repre-senting those crucial pathways deregulated in bladdercancer which can assist in the prediction of nodal metas-tasis. The present study evaluates a panel of 70 transcriptsthat are known to be altered in cancers. The expressionlevels of these genes were determined using StaRT-PCR™and the data was subjected to GP analysis, which identi-fies optimal rules using those genes that it selects as themost significant determinants of the target clinical out-come (in this case, nodal metastasis). StaRT-PCR™ has theability to measure the stoichiometric relationshipbetween the abundance of multiple transcripts within thesame sample [11] and can allow for comparison of datagenerated independently in different experiments and dif-ferent laboratories [19].

Considerations involved in construction of the study cohortThe total study cohort of 65 subjects was divided intotraining and validation sets, and an approximately equaldistribution was attempted between them for each nodalclass within a tumor stage in an effort to eliminate bias[Table 1]. Besides the five normal samples, the rest of thecohort (n = 60) thus has the following distribution: 20NN cases and 3 NP cases in the non-muscularis propria-invasive category (pTa and pT1); and 19 NN cases and 18NP cases in the muscularis propria-invasive category (pT2-4). The cohort thus exhibited an equal proportion of NNand NP cases in the muscularis propria-invasive category,but an unequal proportion of the same in the non-muscu-laris propria-invasive category. These proportions arereflected in the subject distributions in the training andvalidation sets, and may prompt one to surmise that thegene selection process was biased as it recognized tumorstage-specific features rather than those for nodal status.However, given the approximately equitable distributionof NN cases between the non-muscularis propria-invasive

and invasive groups, one can conclude that the featuresidentified by GP corresponded to the absence of nodalmetastasis rather than detrusor muscle invasion or tumorstage. The inequitable distribution of NP cases might leadone to believe that the features identified by GP may cor-respond more to the presence of detrusor muscle invasion(as the number of muscularis propria-invasive cases arehigher) rather than the presence of nodal metastasis. Thiswould, however, mean that GP would not be able to dis-tinguish between NN and NP pTa and pT1 cases, as allthese cases would demonstrate common features of lackof muscle invasion. However, the stage-wise break-up ofthe results show that each time GP was run, it able to dis-tinctly identify between NN and NP pTa and pT1 caseswith 100% accuracy (see Additional file 5). Indeed, thepaucity of NP non-muscularis propria-invasive cases iscommon in clinical settings as only a small minority ofnon-muscularis propria-invasive cases metastasize to thelymph nodes at the time of diagnosis [6]. Alternatively,NP cases are generally considered to be more aggressiveand thus usually present with a greater degree of tumorinvasion. The study cohort was so constructed to reflectthis clinical scenario, albeit using a small number of sub-jects.

The normal subjects were clubbed with the NN cases toconfirm that the technique could recognize normal sam-ples as NN as well. While the genetic makeup of normalurothelium may be entirely different from NN UCs, thecommon theme was the absence of nodal metastasis (andthus, an absence of the genetic features contributing to thesame), rather than the presence or absence of carcinomaafflicting the urothelium. Amalgamation of the twogroups was thus crucial to create a binary classificationsystem that distinguished the presence or absence ofnodal metastasis.

Considerations involved during generation of rulesTranscript levels were used to generate classifier rules thatwere produced after evolving over 100 generations untilthey reached an acceptable level of accuracy. It is necessaryto provide a suitable fitness measure, as fitness is the maindriver of the evolutionary process and thus determines thequality of solutions achieved. One possible measure of fit-ness is a calculation of sensitivity and specificity in a rule'sability to predict the NP subjects. The problem in selectinga single measure of accuracy out of sensitivity and specifi-city is that they are both inherently complementary,wherein increasing one is often associated with decreasingthe other. The overall objective of simultaneously maxi-mizing both parameters was built into the ROC evalua-tion of the test [20], and the search for the mostinformative test sought to maximize the AUC [21]. TheAUC gives a direct indication of how well the samples arebeing separated into different classes and is thus a more



robust fitness measure than any other mathematical com-bination of sensitivity and specificity because there is noconcept of boundary or threshold that can induce discon-tinuities into the system leading to strange behavioraround them.

A major challenge in any machine learning system is toprevent overfitting. This occurs when the function isbiased strongly towards training examples and generalizespoorly to new (unseen) examples. Typically, model over-fitting occurs when there are too few samples relative tothe complexity of the problem. Most clinical problemsmust deal with this issue. In our study, there were only 34samples in the training set from which to learn, with 70variables per sample. This could potentially have led tosolutions that could have been overly biased or overfittedto the training data. This study tried to alleviate the over-fitting problems by restricting the complexity of the resultand by the use of resampling techniques. By restricting thecomplexity of the result, the system was forced to pick outthe most salient features in the data set which were likelyto be the most general solutions [22]. This was achievedby the use of simple mathematical, logical and compari-son operators, and by limiting the size and complexity ofthe programs produced based on the minimum descrip-tion length principle of risk minimization wherein theleast complex solution is called the most robust [23]. Thenumber of genes used in any solution was also restrictedto no more than seven, which puts a constraint on thedegrees of freedom in the expression that is loosely relatedto the VC dimension, a measure of the complexity of aclassification algorithm [24].

For selecting a robust classifier, it is imperative to knowthe generalization rate of the classifier, especially in thecase of small data sets where overfitting can be relativelyfrequent. Cross validation is a resampling technique thatcan help predict the generality of the solution in classifierproblems [25]. In cross validation, the training set is sub-divided into N subsets or "folds" and then each of the N -1 sets are used to learn from and the Nth fold is used to testthe resulting rules. The folds are randomly assembledfrom the whole data set, maintaining the same proportionof true positive cases and true negative cases such thateach fold will have the same representation of samples asthe whole set. To avoid selecting a particularly favorabletest subset, the system is then run again from scratch withanother fold as the test set. This is repeated until all N sub-sets have been used as the test set. The goal is to adjust thesystem so that the results for all training-test set combina-tions (folds) are roughly the same.

While N-fold cross validation is a simple and effectivetechnique to evaluate how well the classifier generalizes tounseen data, the number of folds to use in order to best

assess the general performance of the system is an openquestion. Many machine learning techniques use a leave-one-out cross validation (LOOCV) [26] approach as it hasthe virtue of maximizing the use of samples by allowingthe investigator to view the overall performance of the sys-tem across many folds, suggesting a "normal" behaviorfor the rules generated. LOOCV is approximately unbi-ased for true prediction error, but can have high variancebecause all the "training sets" are similar to each other.This study used a variation of cross validation inspiredfrom the N-fold cross validation scheme that selects anoptimum number of folds that can strike a reasonable bal-ance between bias and variance. Instead of having 34folds, which would correspond to the total number ofsamples in the training set, 11 folds (i.e., the number ofNP samples in the training set) were used that lead to areduction in the variance of the solution. The best per-forming classifiers across all folds were ultimately selectedand applied to a majority voting scheme to generate thebest meta-rule. The majority voting scheme increases theperformance and consistency of the classifiers. It has beenshown that for a binary classification scheme, the per-formance of the aggregate classifier actually increases ifthe individual rules are more than 50% accurate [27,28].Resilience can be significantly improved with thisapproach as estimation errors are reduced.

Clinical relevance of the frequently used genesThe panel of 70 genes was chosen based on an extensivereview of previous studies that have implicated potentialroles for various genes in the progression of cancer in gen-eral and UC in particular. Although many of the candidatemarkers that were selected for this analysis have beenimplicated in one or more of the pathways involved inbladder tumorigenesis, it is plausible that some genes mayplay a relatively more significant role in the developmentof nodal metastasis and the determination of prognosis ifthe disease is detected early. The GP analysis in this studyclearly shows an unequivocal preference to use ICAM1,MAP2K6, KDR, CDK8 and ANXA5 in specific relation-ships to define NP UC specimens. The association of met-astatic disease with the expression levels of these genesand their corresponding proteins is not unreasonable con-sidering their function and involvement in tumor biology.

ICAM1 is a cell surface glycoprotein in the immunoglob-ulin superfamily and is expressed at a low basal level infibroblasts, leukocytes, keratinocytes, endothelial and epi-thelial cells but is upregulated in response to a variety ofinflammatory mediators [29] Several reports indicate thatthe expression levels of ICAM1 correlate with metastaticpotential, migration, and infiltration ability. Immunohis-tochemical studies on 57 patients with bladder carcino-mas revealed that the expression of ICAM1 was closelyassociated with an infiltrative histological phenotype [30]



and serum ICAM1 levels have been related to tumor pres-ence, grade and size in patients with bladder cancer [31].Furthermore, fibrinogen, which may be a determinant ofmetastatic potential [32], mediates bladder cancer cellmigration through an ICAM1-dependent pathway [30].More recently, it was shown that ICAM1 downregulationat the mRNA and protein levels led to a strong suppres-sion of human breast cancer cell invasion through amatrigel matrix and that the level of ICAM1 proteinexpression on the cell surface positively correlated withthe metastatic potential of five human breast cancer celllines [33].

Ligation of ICAM1 induces activation of MAP2K6, whichin turn activates p38 [34-36]. This pathway has beenshown to be closely associated with an invasive pheno-type for bladder tumors [37] and p38 phosphorylation inbreast cancer patients has been associated with a poorprognosis in node-positive tumors [38]. Other studieshave shown a direct effect of MAP2K6 activity on meta-static potential. MAP2K6 transfection into normalMCF10A breast epithelial cells resulted in an invasive andmigratory phenotype accompanied by upregulation ofcertain matrix metalloproteinases [39,40]. Activation ofthis pathway also induced in vitro invasion of normalNIH3T3 fibroblasts [41].

KDR or vascular endothelial growth factor receptor-2(VEGFR2/Flk-1) is a high-affinity plasma membranereceptor for the ligands VEGF-A and -E. This protein isexpressed on endothelial cells in the vasculature andmediates most of the endothelial growth and survival sig-nals through these ligands [42]. Expression of KDR hasalso been demonstrated in tumors of epithelial origin andthe best rules in this study imply that the expression levelof KDR is consistently lower in relation to ICAM1 andMAP2K6 when there is nodal involvement in bladder can-cer. Although a precise reason for why this relationshipshould exist is unknown, some studies have established amore aggressive phenotype in cancers that have lowerexpression of KDR. For example, in patients with UC, highexpression of KDR was associated with increased survivaltimes, whereas those with lower expression values had aworse prognosis [43]. Likewise, high-grade prostate carci-nomas showed much less KDR expression than low ormoderate grade tumors [44]. However, it must be kept inmind that in the present study, it is not the absoluteexpression of KDR that contributes to the correlation withnodal involvement in bladder cancer, but its relationshipto the values of the other two genes.

The identification of CDK8 as a physiological partner ofcyclin C (CycC) is relatively recent [45] and the role of theformer with respect to clinical prognosis in UC has notbeen extensively investigated. The CycC/CDK8 complex is

a part of the pol II holoenzyme complex that plays a partin transcription [46], and is also a part of MED/SRB(Mediator/Suppressor of RNA Polymerase B) containingcomplexes such as TRAP/SMCC (Thyroid hormone-asso-ciated protein/SRB/MED cofactor complexes) and NAT(negative regulator of activated transcription) [47-50].TRAP/SMCC and NAT have been shown to phosphorylateCycH of the general pol II transcription factor, TFIIH com-plex, via their CDK8 kinase activity and inhibit TFIIH pro-tein kinase activity [51]. The suggestion of a lower CDK8level compared to ICAM1 in NP cases through the geneexpression motifs in this study may thus be suggestive ofa role of increased transcription activity, though morefunctional studies in this direction are required.

The annexins are a large family of closely related calcium-and membrane-binding proteins [52] that are expressedin most eukaryotic cell types and appear to participate ina variety of cellular functions including vesicle trafficking,cell division, apoptosis, calcium signaling, and growthregulation. Many of these proteins are differentiallyexpressed in malignant tissue and have been shown to beupregulated or downregulated depending on tumor type[53]. Annexin V (and in the case of our study, annexin A5,encoded by the ANXA5 gene) has been reported to beespecially abundant in platelets where it relocates to thecytoskeleton following stimulus-induced Ca2+ elevation[54,55]. Annexin V is used as a marker for apoptosis [56]and has been shown to influence susceptibility to apopto-sis and pro-inflammatory activities of apoptotic cells [57].Consequently, annexin V expression levels could beaffected by the apoptotic potential of a tumor cell popula-tion, which has been shown to be greatly influenced bythe process of tumor progression and metastasis [58].

Interestingly, previous studies that have attempted toidentify prognostic classes of UC using microarrays havelimited similarity to the gene panel in this study, withE2F4, PCNA, CCNA2 and RB1 being upregulated in highgrade pTa tumors, and ERBB2 being downregulated inmuscularis propria-invasive tumors [59]. However, onemust note that such studies generally consider tumorgrade and stage as prognostic indicators, and their molec-ular signatures are usually filtered to reflect the same. Onthe other hand, our study defined nodal metastasis as aprognosticator and the GP system was thus trained toidentify genetic traits that best corresponded to this indi-cator. Furthermore, the identification of a single ruleemploying the three most frequently used genes proved tobe more robust in terms of predicting the nodal stage inthe validation set. While this study suggests a possible piv-otal role of the expression of these three genes in deter-mining nodal status, the limitation of a relatively smallsample size compared to the number of variablesinvolved warrants similar studies with larger sample sizes



to validate these results. One can hypothesize that com-parative downregulation of KDR (and perhaps, angiogen-esis in general) in the primary tumor might impartselection pressure for invasion leading to upregulation ofICAM1 that reflects a tumor's potential to establish a met-astatic lesion in a draining lymph node. This possiblemechanism can partially explain the gene transitivityobserved, whereby the gene expression signature of nodalmetastatic cases consistently conforms to a fixed motif ofICAM1>MAP2K6>KDR, although a literature search didnot reveal any study that investigated these genes in con-cert in the context of nodal metastasis. The above motifalso exemplifies the hypothesis-generating nature of GP,and directed investigations into the role of these genesand their respective pathways in promoting nodal metas-tasis are required. Further work is necessary to show thatthis approach is effective in all cases but it tends to sup-port the theory put forth by Daida that GP has two phases:identifying the key inputs, and then finding rules thatoptimally combine them [60]. By using specific genesshown to be more useful during pilot studies, it allows theGP process to search for the best use of those genes. Thiscan enable the GP system to accurately predict prognosisand help in making therapeutic decisions, thus having adirect impact on the patient and the treating clinician.

Advantages of genetic programmingAs can be seen from the results, GP is distinct from othercommon machine learning algorithms used in bioinfor-matics [Table 6]. This technique is gradually gaining pop-ularity for the analysis of medical and biological data, andfor the prognostic classification of cancers [61-65].

A unique feature of GP is the final output, which consistsof easily readable rules expressed as executable classifierprograms that define tangible relationships between themost influential genes. This allows the results to be putinto the known biological context of these genes, whichcan enhance their significance or provide new workinghypotheses that could be further tested. Most other classi-fier algorithms like Support Vector Machines (SVM), Neu-ral Networks and K-Nearest Neighbors (KNN) clusteringapproaches do not provide human readable results. Hier-archical clustering creates visually intuitive results but the

output does not specify an exact relationship amongst thegenes. Classification and regression trees (CART) output abinary decision tree that comes closest to GP developedrules in terms of human readability but fail to provideclear insights into gene co-expressions that become moredifficult to discern due to the relationships becoming lessexplicit as the trees grow larger. While CART algorithmsare normally felt to be greedy, aiming to locally optimizethe decision tree during construction of the solution [66],GP takes on a more global view of the solution space andcan thus search a larger space for solution trees that mightlead to improved performance.

By identifying those genes that are most dominant indefining outcome, the GP process can usually limit thecomplexity of the classifiers and generate robust but sim-ple rules containing only a few genes without compromis-ing their predictability. This is potentially useful in aclinical setting where profiling each gene has its own cost.The smaller the gene set needed to make a clinical diagno-sis, the cheaper the test and potentially the more accepta-ble it is to payors.

GP does not assume extensive prior knowledge on theexpected form of the solution or any preconceived geneticinteractions to set up the analysis. This is especially usefulgiven that genetic relationships are not always wellknown. Hence, one can judge how it could be difficult touse classification or clustering algorithms where oneneeds to pre-specify the structure of the expected solution.For example, SVMs involve selecting a kernel for mappingthe data to a higher dimensional space, which is non-triv-ial and often a non-intuitive process that can affect theaccuracy of the classifiers.

GP can also select variables automatically without anyneed to pre-filter or limit them based on what is knownabout a system. Such filtering is usually done because ofthe combinatorial problem of working with a largenumber of inputs; however, such filtering can create anincomplete and biased dataset that may not be represent-ative of many complex biological systems. The "curse ofdimensionality" [67] affects all classification algorithmsbut the problem of dimensionality reduction is more

Table 6: Advantages of genetic programming.

Method Human Readability Automatic Selection of Variables

Automatic Integration of Data Types

Non-Linear Relationships

Statistical Analysis Yes Limited No LimitedCluster Analysis Yes No No No

Support Vector Machine No No No YesNeural Networks No No No Yes

Genetic Programming Yes Yes Yes Yes



important in classical algorithms like hierarchical, KNN,K-means clustering and Neural Nets which do not scaleeasily to larger numbers of variables. Feature selection isthen an important step before the application of thesealgorithms and can lead to loss of information that mightbe critical for the success of the learning algorithm.

As can be seen from the rules generated in our study, mostrules express non-linear relationships such as MAP2K6/KDR or ICAM1/CDK8. The ability to choose variablesfrom a large list and then combine them in a non-linear,readable way is a powerful feature of this approach asmany biological systems often have non-linear relation-ships between genes or proteins. SVM is a popular algo-rithm which outputs non-linear classifiers but is limitedby the kernel selected. CART algorithms implement non-linearity in a pseudo sense as they split the data and tackleeach partition separately, but are not as succinct as therules produced by GP in capturing the effects and the rela-tionships among gene expressions.

Lastly, GP can incorporate very diverse data sets that con-tain markedly different types of variables and can alsohandle missing values in the data. Missing data can be animportant problem as even a small amount of missingdata can lead to a large loss in performance. This is espe-cially true in systems like Hierarchical or KNN based clus-tering and SVMs, that necessitate the use of various toolslike imputation, replacement of the missing values with aconstant, and removal of samples with a large amount ofmissing data. However, most of these approaches canintroduce bias in the system due to the assumptions madeabout the missing data that could lead to loss of impor-tant features. GP alleviates this problem by leveraging theability of the system to automatically select features.Whenever a rule encounters missing data in a sample dur-ing fitness assessment, the sample is labeled as misclassi-fied, thus decreasing the fitness of the rule. Thus, thesystem is not favorably disposed towards picking up a var-iable (feature) that is laden with a large percentage ofmissing values. This approach allows for maximum use ofthe available data without making any unwarrantedassumptions about missing data.

Limitations of genetic programmingGP is a computationally intense process requiring a largeamount of machine time. The estimated machine timeincreases with increasing complexity of the problem, andincrease in the dimensions and number of samples. Thiscan be resolved by using parallel processing and segment-ing the problem into parts which can be performed on dif-ferent processors simultaneously and then synchronizingamong them. GP is particularly tractable for parallel com-puting techniques as there are several natural ways to dis-tribute execution onto different machines [68].

As GP is a stochastic process that depends highly on theinitial control parameter settings, it does not guarantee anoptimal solution in all runs. It should therefore be runseveral times with different settings to ensure that the sys-tem has not fallen into a local optima.

While GP combines features of global and local searchalgorithms, the cost is that it often performs neither ofthese functions as well as more specialized algorithms.The constant introduction of new genetic materialthrough mechanisms of mutation and crossover (mating)will divert the algorithm from finding the best combina-tion of a few highly effective components. For this reason,this study adopted a two-phase strategy where the mostimportant variables were identified from the list of themost frequently chosen variables [Figure 3] and the sys-tem was then run again using only those high frequencyvariables. The first pass allowed the GP system to globallysearch a large set of possible variable combinations whilethe second pass let it locally search for the best combina-tion of those variables.

GP may also output several rules that are quite differentbut perform equally well, thus suggesting the involvementof multiple and often unrelated genes. The selection of asingle rule can be difficult, particularly when searching fora general solution to a problem. This led us to adopt thevoting algorithm to tackle the problem of rule selectionand consistency. It would also seem logical to be relativelysure of the biological functionality of the genes selectedunless there is sufficient data to confirm an unusual ruleor gene selection. This is not a limitation of GP per se, butrather a limitation of any machine learning algorithm.

ConclusionOur study uses UC as a clinical model in devising a strat-egy to combine the medium-throughput quantitativeStaRT-PCR™ technique with supervised GP methods todetermine the nodal status of clinically diagnosed tumorsbased on their molecular profiles. We demonstrate thatStaRT-PCR™ can provide a relatively standardized outputof quantitative gene expression relative to a housekeepinggene like β-actin and can be used as an input in a GP sys-tem to generate a classifier for nodal status with a reason-able degree of accuracy. Moreover, the output has alsosuggested a key role for specific genes involved in the tar-get process that may lead to future studies to clarify theirprecise biological role and identify new targets for thera-peutic intervention. Of particular interest are the geneexpression motifs which have identified novel relation-ships between specific genes and pathways. The key genesidentified by this technique from our data set also suggestthat class-specific signatures using a small number ofgenes can characterize tumors as NP or NN, and more



importantly, provide an early indication of their progres-sion towards NP status based on molecular traits.

Our group is currently addressing several open questionsin GP including an approach for multi-class problems,automated methods for selecting key transcripts and auto-mated identification of significant motifs. Further studieswill be aimed at correlating molecular markers and motifswith clinical outcome in an effort to employ markers asreliable, reproducible and objective indicators of progno-sis. The enhanced value of incorporating molecular mark-ers into the existing clinical staging of bladder cancer hasalready been proposed as a prudent alternative [69]. GPwill then be ultimately useful in the identification of newavenues of molecular investigations, critical componentsand signatures of prognosis, and therapeutically feasibletargets.

AbbreviationsUC → urothelial carcinoma

StaRT-PCR™ → standardized competitive reverse tran-scriptase – polymerase chain reaction

GP → genetic programming

NP → node positive

NN → node negative

CT → competitive template

AUC → area under curve

ROC → receiver operating characteristic

KDR → kinase insert domain receptor

MAP2K6 → mitogen-activated protein kinase kinase 6

ICAM1 → intercellular adhesion molecule 1

CDK8 → cyclin-dependent kinase 8

ANXA5 → annexin A5 (gene)

LOOCV → leave-one-out cross validation

VEGF → vascular endothelial growth factor

CycC → cyclin C

MED/SRB → mediator/suppressor of RNA polymerase B

TRAP/SMCC → thyroid hormone-associated protein/SRB/MED cofactor complexes

NAT → negative regulator of activated transcription

SVM → support vector machines

KNN → K-Nearest Neighbors

CART → classification and regression trees

Competing interestsAuthors AAA, DWF, PFL and WPW are employees ofGenetics Squared Inc. (Ann Arbor, MI, USA). RJC is amember of the scientific advisory board for GeneticsSquared Inc., which is developing the commercial uses ofthe genetic programming system used in this study. Otherauthors declare no competing interests.

Authors' contributionsAPM was responsible for organization of the studycohorts, genetic programming outcome analysis, investi-gation of molecular biology and clinico-pathological cor-relations and co-drafting the manuscript. AAA wasresponsible for genetic programming analysis of the dataand co-drafting the manuscript. BG and VP carried out theStaRT-PCR™ experiments. DWF researched and co-authored the section on the clinical relevance of the fre-quently used genes and critically revised the manuscript.PFL provided background clinical information on bladdercancer, and the rationale and potential significance of thestudy from a clinical oncology perspective, and criticallyrevised the manuscript. RJC and RHD were responsiblefor designing the StaRT-PCR™ experiments, and partici-pated in the design and coordination of the study. WPWdirected the computational part of this study, drafted por-tions of the computing sections of the manuscript andparticipated in overall editing. All authors read andapproved the final manuscript.

Additional material

Additional file 1A comprehensive list of the genes investigated using StaRT-PCR™ includ-ing their GenBank accession, GeneID and UniGene cluster numbers.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2407-6-159-S1.pdf]

Additional file 2The box plots for the gene expression values for KDR, MAP2K6 and ICAM1 that are differentially expressed in the primary tumor tissues of the node positive and the node negative classes.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2407-6-159-S2.pdf]


http://www.biomedcentral.com/content/supplementary/1471-2407-6-159-S1.pdf



AcknowledgementsThe authors would like to thank Frederic Waldman, MD, PhD, for provid-ing the RNA samples from 10 subjects who underwent treatment for pTa and pT1 bladder cancer at the University of California, San Francisco, USA. The molecular studies of bladder cancer progression were funded by National Institutes of Health Grants CA-65726, CA-70903 and CA-86871.

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Additional file 3The classifier rules that form the meta-rule generated for node positive patients using the three most frequently used genes, viz. KDR, MAP2K6 and ICAM1.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2407-6-159-S3.pdf]

Additional file 4The performance metrics of the first classifier rule from the meta-rule gen-erated for node positive patients using the three most frequently used genes, viz. KDR, MAP2K6 and ICAM1, on the validation set.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2407-6-159-S4.pdf]

Additional file 5The stage-wise and case-wise performance of the meta-rules generated using 70 genes and the three most frequently used genes, viz. KDR, MAP2K6 and ICAM1, on the validation set. This file also shows the stage- and case-wise performance of the single rule "(MAP2K6/KDR) × (1.0 - (MAP2K6/ICAM1)) > .71" selected from the meta-rule generated using the three most frequently used genes on the validation set.Click here for file[http://www.biomedcentral.com/content/supplementary/1471-2407-6-159-S5.pdf]





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