Intratumoural cytogenetic heterogeneity of sporadic colorectal carcinomas suggests several pathways...

ORIGINAL PAPERJournal of PathologyJ Pathol 2010; 221: 308–319Published online 23 April 2010 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/path.2712

Intratumoural cytogenetic heterogeneity of sporadic colorectalcarcinomas suggests several pathways to liver metastasisJose Marıa Sayagues,1 Marıa del Mar Abad,2 Hermann Barquero Melchor,3 Marıa Laura Gutierrez,1Marıa Gonzalez-Gonzalez,1 Evan Jensen,1 Oscar Bengoechea,2 Emilio Fonseca,4 Alberto Orfao1*†

and Luıs Munoz-Bellvis3†

1 Servicio General de Citometr ıa, Departamento de Medicina and Centro de Investigacion del Cancer (IBMCC-CSIC/USAL), Universidad deSalamanca, Salamanca, Spain2 Departamento de Patolog ıa, Hospital Universitario de Salamanca, Salamanca, Spain3 Unidad de Cirug ıa Hepatobiliopancreatica, Departamento de Cirug ıa, Hospital Universitario de Salamanca, Salamanca, Spain4 Servicio de Oncolog ıa Medica, Hospital Universitario de Salamanca, Salamanca, Spain

*Correspondence to: Professor Alberto Orfao, Centro de Investigacion del Cancer, Paseo de la Universidad de Coimbra S/N, 37007Salamanca, Spain. e-mail: [email protected]

†These authors contributed equally to this work and should be considered as senior last authors.

AbstractMuch has been learned about the chromosomal abnormalities of colorectal carcinomas but the cytogeneticrelationship between the neoplastic clones present in primary versus metastatic tumour samples remains unclear.We analyse the frequency of abnormalities for 47 chromosome regions using the interphase fluorescence in situhybridization technique in a group of 48 tumours, including 24 primary colorectal tumours and 24 pairedliver metastases. All tumours showed complex karyotypes with numerical/structural abnormalities for seven ormore different chromosomes/chromosome regions both in the primary tumours and in their paired metastases.Chromosome 8 was the most frequently altered (22/24 primary tumours), consistently showing del(8p22) and/orgains/amplification of 8q24, followed by abnormalities of the entire chromosome 7 (21/24 primary tumours) andof chromosomes 17p and 20q (20/24 primary tumours). Simultaneous staining for multiple chromosome probesrevealed the presence of two or more tumour cell clones in 23/24 cases (46/48 tumour samples). Interestingly, theliver metastases typically contained tumour cell clones similar to those found in the primary tumours, suggestingthe absence of selective selection of specific tumour clones. Despite this, additional chromosomal abnormalitieswere detected in 23/24 metastatic tumours, which preferentially consisted of del(17p13) and gains/amplificationof 11q13 and 20q13; moreover, compared to primary tumours, metastases showed an increased numberof abnormalities of chromosomes 1p, 7q, 8q, 13q, and 18q, and new chromosomal abnormalities involvingchromosomes 6, 10q23, 14q32, 15q22, and 19q13. Owing to the high frequency of numerical abnormalities ofthe entire chromosome 7 and loss and/or gain/amplification of specific regions of chromosome 8, eg del(8p22)and/or gains/amplification of 8q24 in primary colorectal tumours with associated metastases, it is suggested thattheir assessment at diagnosis could be of great clinical utility for the identification of colorectal cancer patientsat higher risk of developing liver metastases. Copyright 2010 Pathological Society of Great Britain and Ireland. Publishedby John Wiley & Sons, Ltd.

Keywords: liver metastases; colorectal carcinoma; chromosomal abnormalities; iFISH; clonal evolution; histopathology

Received 23 October 2009; Revised 10 March 2010; Accepted 15 March 2010

No conflicts of interest were declared.

Introduction

Colorectal cancer (CRC) is one of the most com-mon malignant tumours and is frequently lethal due tometastatic spreading to the liver [1,2]. To date, mostgenetic studies on CRC have focused on the earlychanges responsible for the initial oncogenic events.Thus, it is now well established that the develop-ment and progression of CRC are associated with anaccumulation of chromosomal abnormalities involv-ing key regulatory genes in normal colonic mucosal

cells [3–6]. Among other abnormalities, CRC has beenassociated with mutations involving (i) the adenoma-tous polyposis coli (APC ) gene, which is thought tooccur early on during the development of adenomatouspolyps; (ii) activation of the RAS oncogene (duringthe adenomatous stage); and (iii) concurrent deletion ofmultiple chromosomal regions in the transition to car-cinoma (ie those involving chromosomes 17p and 18q,which contain the TP53 and DCC genes, respectively),in addition to genes involved in DNA repair [7–11].In contrast, chromosomal abnormalities involved in

Copyright 2010 Pathological Society of Great Britain and Ireland. J Pathol 2010; 221: 308–319Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com

Cytogenetic heterogeneity of primary versus metastatic colorectal cancer 309

determining disease progression to a metastatic tumourremain largely unclear.

Recent years have witnessed an increase in infor-mation about the specific chromosomal abnormalitiesof primary CRC as detected by conventional cytoge-netics and comparative genomic hybridization (CGH),among other techniques. Such studies have revealeda non-random pattern of genomic abnormalities in pri-mary carcinomas, which frequently include gains of the7p, 8q, 13q, and 20q chromosomal regions and lossesof 1p, 4, 5q, 8p, 9q, 14q, 17p, and 18q [12–22]. How-ever, although metastases are usually the ultimate causeof death in CRC patients, the chromosomal abnor-malities characteristic of such advanced stages of thedisease are complex and have been poorly described[23,24]. In addition, most studies have used either con-ventional cytogenetics or molecular approaches suchas CGH-array techniques to define the chromosomaland molecular abnormalities of CRC, despite the factthat they both have important limitations: they alloweither the analysis of only a small part of all tumourcells, or the detection of gains or losses of geneticmaterial present in the majority (eg more than 60%)of cells in the tumour sample from which DNA wasextracted [25,26]. In addition, such approaches do notprovide detailed information about the clonal hetero-geneity and genetic diversity of a given tumour andthe potential co-existence of different abnormalities inthe same tumour sample, particularly when tumour cellclones are present at relatively low frequencies [26,27].Because of this, information about the precise pat-terns of intratumoural clonal evolution and the relation-ship between the neoplastic clones present in primaryversus metastatic tumour samples remains very lim-ited [12,28]. Although interphase fluorescence in situhybridization (iFISH) does not provide specific infor-mation about each chromosomal abnormality present ina tumour, it may overcome some of the limitations ofconventional cytogenetics and CGH techniques, as longas an adequate combination of probes is used to obtaininformation about the genetic diversity of a tumour atthe single-cell level [29].

Here, we applied multi-colour iFISH for the analysisof 47 different DNA sequences distributed acrossthe most frequently altered chromosomes in bothprimary tumours and their paired liver metastasesfrom 24 untreated CRC patients. Our major goalwas to establish the intratumoural pathways of clonalevolution associated with chromosomal instability inindividual primary versus metastatic tumours fromcolorectal carcinoma patients.

Materials and methods

Patients and samplesTissue specimens from 24 sporadic colorectal ade-nocarcinomas and 24 paired liver metastases (n =48 samples) were obtained from the same number

of patients (16 males and eight females) before anysystemic treatment or local radiotherapy was given.The median age was 68 years (range 48–80 years).All patients had undergone surgical resection of bothtumour tissues at the Department of Surgery of theUniversity Hospital of Salamanca (Salamanca, Spain)between January 2001 and August 2007, and theywere recruited into the study after they had given theirinformed consent to participate. The study underwentinstitutional review and was approved by the LocalEthics Committee of the University Hospital of Sala-manca (Salamanca, Spain). Salient clinical and labo-ratory data of the 24 patients studied are summarizedin Table 1 and are fully described in the Supportinginformation, Supplementary methods.

Interphase fluorescence in situ hybridization (iFISH)studiesIn all tumours, a mixed single-cell suspension fromthe different samples obtained from each tumourwas used for the iFISH studies after fixation in 3/1methanol/acetic (vol/vol). A set of 47 different probesspecific for those chromosomes and chromosomalregions most frequently gained/amplified and deletedin sporadic colorectal carcinomas [5,30–33] was sys-tematically used in triple staining (Figure 1). In orderto define accurately the exact abnormalities carried bya tumour cell clone within a tumour sample, furtherappropriate multi-colour staining was performed whennecessary. The methods and procedures used for theiFISH studies have been previously described in detail[29] and detailed information is included in the Sup-porting information, Supplementary methods.

Quantification of the number of hybridization spotsper nuclei was performed using a BX60 fluorescencemicroscope (Olympus, Hamburg, Germany) equippedwith a 100× oil objective for ≥ 200 cells per sam-ple. Only spots of similar size, intensity, and shapewere counted in areas with less than 1% non-hybridizedcells; doublet signals were considered as single spots.A tumour was considered to carry numerical abnor-malities for a given chromosome region when theproportion of cells displaying an abnormal number ofhybridization spots for the corresponding probe wasat a percentage higher or lower than the mean valueplus two standard deviations (SD) of the percentageobtained with the same probe in normal control periph-eral blood samples (n = 10) (Figure 2). In turn, thedefinition of a tumour cell clone carrying a chromo-somal abnormality was based on the presence of morethan 5% nuclei bearing identical numbers of hybridiza-tion spots for all probes analysed, including the alteredones. Hereafter, the term ‘numerical abnormality’ isused to describe changes that involve an entire chro-mosome consisting of either gain or loss of thechromosome; in turn, the term ‘structural abnormality’is employed when referring to chromosomal abnormal-ities which only involve a chromosome arm or chromo-some region, indicating that the chromosome structure


310 JM Sayagues et al

Table 1. Clinical and biological characteristics of the colorectal cancer patients with paired liver metastases analysed (n = 24)

No of tumoural cell clones by iFISH

Site of primary Histological

Case ID Gender Age (years) tumour grade TNM stage Primary tumour Liver metastasis

1 M 74 Right colon Well T3N2M1 3 32 M 60 Right colon Well T3N0M1 2 23 M 77 Left colon Well T3N1M1 2 24 F 80 Left colon Well T4N0M1 3 25 M 64 Left colon Well T3N0M1 3 26 F 51 Left colon Well T3N2M1 2 27 M 69 Left colon Well T3N0M1 3 28 M 57 Left colon Well T3N1M1 2 29 F 67 Left colon Well T3N1M1 2 3

10 M 79 Rectum Well T4N0M1 2 211 M 63 Rectum Well T3N2M1 2 312 M 77 Rectum Well T3N1M1 2 213 M 63 Rectum Well T4N0M1 2 114 M 75 Rectum Well T3N1M1 3 215 F 76 Rectum Moderate T3N1M1 2 216 M 49 Rectum Moderate T3N1M1 1 217 M 74 Rectum Moderate T4N0M1 2 218 M 62 Right colon Moderate T3N2M1 2 219 F 76 Right colon Moderate T3N1M1 2 220 F 58 Left colon Moderate T4N2M1 2 321 F 79 Left colon Moderate T4N0M1 2 222 F 75 Left colon Moderate T4N1M1 2 123 F 48 Left colon Poor T4N2M1 2 324 M 72 Left colon Poor T3N1M1 2 2

M = male; F = female; iFISH = interphase fluorescence in situ hybridization; well = well-differentiated adenocarcinoma; moderate = moderately differentiatedadenocarcinoma; poor = poorly differentiated adenocarcinoma.

is altered. Detailed information pertinent to statisticalstudies can be found in the Supporting information,Supplementary methods.

Results

Frequency and type of chromosomal abnormalitiesin primary CRCs versus their paired liver metastasesFrom the cytogenetic point of view, all CRC tumours(n = 48) analysed showed complex karyotypes withnumerical and/or structural abnormalities involvingseven or more chromosomes in both primary (n = 24)and metastatic tumours (n = 24). Overall, chromo-some gains were found more frequently than chro-mosome losses, both in primary tumours and in theirmetastases (55% versus 11% and 63% versus 10% ofall chromosome abnormalities detected, respectively),and a majority of all gains reflected the existence ofpolyploid karyotypes. Considering all chromosomestogether, chromosome 8 was the one most frequentlyaltered in primary tumours (22/24 tumours), followedby abnormalities involving the entire chromosome 7(21/24 tumours) and the 17p and 20q chromosomearms (20/24 tumours). Regarding chromosome gains,disomy X in males (69% of tumours) and gains ofchromosome 20 (67% of tumours: trisomy in 4% of thetumours; tetrasomy in 13%; pentasomy in 29%; hex-asomy in 17%; and octosomy in 4% of the tumours)were the most frequent abnormalities; in turn, the most

frequently detected chromosome losses correspondedto monosomy 18 (33%) and nulisomy Y in males (31%of tumours). Losses at chromosomes 1p (46%), 8p[58% of tumours: 16% with del(8p) alone and 42%with combined del(8p) and 8q+], and 17p (66%), andgains/amplification at 8q [54%: 12% with 8q+ aloneand 42% with 8q+ in association with del(8p)] werethe structural abnormalities most frequently detectedand they were frequently associated with hyperdiploidkaryotypes (29%, 41%, 37%, and 33% of tumours,respectively).

Overall, the chromosomal abnormalities identifiedin liver metastases showed profiles similar to thosefound in primary tumours, although the frequencyof each individual aberration was slightly different.Thus, gains/amplification at 14q32 were detected in58% of metastatic versus 33% of primary tumours(p < 0.001); similarly, gains/amplification of chromo-somes 10q23 (found in 50% of the metastases versus42% of all primary tumours; p = 0.003), 6p11.1 (67%versus 54%; p < 0.001), 6q21 (63% versus 46%; p =0.001), 6q23 (67% versus 54%; p = 0.001), 15q22(54% versus 42%; p < 0.001), and 19q13 (75% ver-sus 58%; p = 0.002) were more frequently identifiedin metastatic than in primary tumours, respectively.Table 2 and Figure 3 summarize the specific abnormal-ities detected for each chromosome both in primarytumours and in their liver metastases (see Support-ing information, Supplementary Table 1 for a moredetailed description of such alterations).



Figure 1. Chromosome distribution of the iFISH probes directed against 47 different specific genes, loci, and centromeric regions of the24 human chromosomes, used in the present study. Green, red, and blue lines correspond to the Spectrum Green, Spectrum Orange, andAqua fluorochromes conjugated to the probes, respectively. All probes were purchased from Vysis Inc (Chicago, IL, USA), except for the3q26, 6q21, 11p11.1, 11q13, 12p11.1, 12q13, 15p11.1, 15p22, and 19q13 probes, which were obtained from Q-BIOgene Inc (Amsterdam,The Netherlands).

Figure 2. Representative pictures of cell nuclei from normalcontrol peripheral blood samples (n = 10) after hybridization witha probe for chromosomes 8p22 (red spots), 8p11 (blue spots), and8q24 (green spots). All cell nuclei showed a normal diploid numberof hybridization spots for the three chromosome probes studied.

Cytogenetic associations in primary tumours versuspaired liver metastases

Upon analysing the potential existence of an associa-tion between the number of iFISH signals detected foreach chromosome probe in primary colorectal tumours

Figure 3. Summary results highlighting the frequency of thosechromosome gains more frequently identified in primary tumours(PT) and their paired liver metastases (LM). The changes identifiedfor these chromosome regions mainly consisted of an alterednumber of copies detected by iFISH (see Table 5).

(Table 3), a remarkably significant association (r2 ≥0.75; p < 0.001) was found among gains/amplificationof the 6q, 11q13, and 13q34 chromosomal regions.In addition, chromosomal abnormalities involving the9q34, 10q23, 12q13, 13q, and 22q11 regions were alsoassociated with gains/amplification of 7q31 (r2 ≥ 0.70;p < 0.001); in turn, losses in the 18q21 chromosomeregion were related to del(17p13) and structural abnor-malities of chromosome 12q13 (r2 ≥ 0.73; p < 0.001).In addition, a significant association was also foundamong 13q34 gains and numerical abnormalities ofchromosome 5 (trisomy and tetrasomy) (r2 = 0.82;p < 0.001).



Tabl

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abno

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and

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tum

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and

thei

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liver

met

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case

sw

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tere

dch

rom

osom

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mou

r/liv

erm

etas

tase

s)

12

34

56

710

1112

1314

1516

1718

1920

2122

XfXm

Y

Nor

mal

29/2

529

/29

25/2

142

/42

33/2

929

/25

13/1

28/

825

/21

58/3

825

/25

30/2

521

/13

50/2

546

/38

41/3

317

/13

38/3

342

/25

17/1

742

/46

67/6

350

/50

31/1

925

/12

Gai

ns p+− /

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−8/

4−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−q+

−/−

−/−

26/2

5−/

−−/

84/

44/

412

/12

8/8

−/−

17∗ /3

4∗−/

−20

/24

−/−

21/2

9−/

−−/

−−/

−−/

−16

/16

−/−

−/−

−/−

−/−

−/−

Poly

som

y25

/29

67/7

149

/54

50/5

034

/21

38/5

062

/59

22/2

254

/50

42/5

038

/33

50/5

959

/59

33/5

821

/25

46/5

48/

25−/

458

/75

67/6

750

/46

20/2

450

/50

69/8

144

/50

Loss

es

Nul

isom

y−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−31

/38

Mon

osom

y−/

4−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

4−/

−−/

−−/

−17

/17

4/4

13/1

39/

933

/29

−/−

−/−

8/8

13/1

3−/

−−/

−−/

−de

l(p)

46/4

24/

−−/

−8/

8−/

−−/

−−/

−16

/20

−/−

−/−

8/4

8/8

−/−

−/−

−/−

−/−

66/5

3−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−de

l(q)

−/−

−/−

−/−

−/−

33/4

229

/21

21/2

5−/

−9/

21−/

812

/44/

4−/

4−/

−8/

4−/

−−/

−29

/34

−/−

−/−

−/−

−/−

−/−

−/−

−/−

del(p

)/q+

−/−

−/−

−/−

−/−

−/−

−/−

−/−

42/3

84/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−−/

−

89

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Regarding liver metastases (Table 3), a significantcorrelation was observed between gains/amplificationof chromosome 20q13 and losses in the 18q21 and17p13 chromosomal regions (r2 ≥ 0.70; p < 0.001),the latter also being associated with gains/amplificationof 6q23 and 13q (r2 ≥ 0.73; p < 0.001). Similarly, asignificant association was found between the abnor-malities involving the 9q34 and 12p13 chromosomeregions (r2 = 0.71; p < 0.001). Other significant cor-relations detected among the different chromosomalabnormalities found in liver metastases could beexplained in terms of probes that map to the samechromosome (6q21 and 6q23, r2 = 0.91; 12p13 and12q13, r2 = 0.80; 13q14 and 13q34, r2 = 0.88), chro-mosome regions that were not altered (1p36 and 15q22,r2 = 0.71), and gains derived from chromosomal insta-bility associated with triploid and tetraploid karyotypes(1q25 and 10q23, r2 = 0.80; 2p24 and 6q23, r2 =0.73; 2p24 and 9q34, r2 = 0.73; 2p24 and 10q23,r2 = 0.80; 2p24 and 12q13, r2 = 0.73; 2p24 and14q32, r2 = 0.73; 12q13 and 14q32, r2 = 0.79; 12q13and 19q13, r2 = 0.70; 15q22 and 19q13, r2 = 0.70).Interestingly, gains/amplification of chromosome 8q24showed no statistically significant associations withany other chromosomal abnormality, either in primarytumours or in their liver metastases. Of note, no signif-icant association was found between individual chro-mosomal abnormalities and age, tumour localization,or other characteristics of the disease at diagnosis.

Intratumoural patterns of chromosomalabnormalitiesSimultaneous iFISH staining for multiple chromo-some probes of both primary CRC tumours and theirpaired liver metastases revealed the presence of twoor more tumour cell clones in 18 (75%) and 5 (21%)tumours, respectively; in each tumour, each of theclones represented ≥ 7% of all cells in the sam-ple. Supplementary Table 2 in the Supporting infor-mation offers a detailed description of the differenttumour cell clones present in each primary tumourversus its paired liver metastasis. Detailed analysis ofthe chromosomal abnormalities detected in the dif-ferent tumour cell clones identified in each tumoursample allowed us to speculate about the underly-ing patterns of intratumoural clonal evolution. Clonescontaining chromosomal abnormalities that were com-mon to all tumour cells in a sample were consid-ered to be the ancestral tumour cell clone; in turn,those containing alterations which involved only aproportion of the tumour cells were considered sec-ondary clones. Figure 4 summarizes the hypotheticalpathways of intratumoural clonal evolution detectedfor the most frequently altered chromosomes. Asshown, the presence of a tumour cell clone withlosses in the 8p, 17p, and 18q chromosomal regionsand gains/amplification in 8q and 20q (associated ornot with gains of chromosomes 11 and 13q) wasthe early cytogenetic profile most frequently detected

(n = 8; 34%). Interestingly, liver metastases typicallyshowed the same tumoural cell clones as those foundin their paired primary tumours, although additionalclones acquiring new chromosomal abnormalities weredetected in at least one of the clones in 23/24 tumours(Figure 4), differences consisting in a greater num-ber of copies of specific chromosomal regions (8q24,11q13, 13q34, and 20q13) in metastatic versus pri-mary tumours. In addition, new abnormalities involv-ing chromosomes 14q32, 10q23, 6, and 19q13 werealso detected in metastatic samples, although at lowerfrequencies.

As shown in Figure 5 for tumour 18, metastatictumour cells from this patient acquired additionalabnormalities for chromosomes 8q24 (3 versus 8copies), 11q13 (2 versus 8 copies), and 20q13 (5 ver-sus 10 copies) compared with the primary tumour. Infact, a significant correlation was observed betweenthe number of signals obtained by iFISH for eachindividual probe used in primary colorectal tumoursand their paired metastases, particularly for chromo-somes 2p, 4p, X in females and Y in males (r2 ≥ 0.89)(Table 4). Nevertheless, this correlation was relativelypoor for some chromosomal regions, especially forchromosomes 11q13 (r2 = 0.40), 17p13 (r2 = 0.43),and 20q13.2 (r2 = 0.44), with a higher frequency ofmetastatic tumours (9/24) showing additional 11q13and 20q13 abnormalities among liver metastases versusprimary tumours. The specific chromosomal abnormal-ities detected for these and other chromosome regionsbetween primary colorectal tumours and their pairedliver metastases are shown in Table 5. Interestingly,those chromosomes most frequently altered in livermetastases (versus a normal diploid profile in pri-mary tumours) showed intermediate correlation coef-ficients: 14q32 (r2 = 0.80), 10q23 (r2 = 0.60), 6p11.1(r2 = 0.74), 6q21 (r2 = 0.65), 6q23 (r2 = 0.69) 15q22(r2 = 0.83), 19q13 (r2 = 0.74), and 8p22 (r2 = 0.69)(Table 4).

Discussion

Early studies by Vogelstein and Fearon [3,34] identi-fied recurrent genetic markers and profiles in colorectaltumours whose frequency varied from adenomas to col-orectal carcinomas. Among other abnormalities, thesemainly included losses of the 18q, 17p, and 5q chro-mosome regions, involving the DCC, TP53, and APCgenes, in addition to KRAS mutations. Based on thedistinct cytogenetic profiles of different histologicallybenign versus malignant colorectal tumours, a widelyaccepted model of multi-step colorectal tumourigenesishas been proposed [34]. In this model, it is assumedthat 5q losses could represent an initial event in thetransformation from normal epithelial cells to early-stage adenomas; subsequent loss of 18q would charac-terize further progression into the adenomatous polypstage and finally loss of chromosome 17p would corre-late with transition to carcinoma. Despite its relevance,



Table 3. Correlation between the number of iFISH signals detected for each pair of chromosome probes in primary colorectal tumours(n = 24) and liver metastases (n = 24)

Results reported as Pearson correlation coefficients (R2); empty spaces correspond to those correlations between chromosome regions which showed a correlationcoefficient (R2) less than 0.70.

this model has not been validated at the intratumouralcell level and it only provides limited information con-cerning the genetic changes that could be invoked toexplain the occurrence of distant metastases. In addi-tion, several frequently detected chromosomal abnor-malities, such as those involving losses of chromo-somes 1p and 8p and gains/amplification of chromo-somes 8q, 11q, 13q, and 20q [34], are not taken intoaccount in the model.

Cytogenetic profiles of colorectal cancer metastases

Previous studies have consistently identified a higherfrequency of liver metastases in CRC tumours dis-playing complex karyotypes (tumours with more thanthree chromosomal abnormalities) bearing a loss ofheterozygosity in chromosomes 5q [35,36], 14q [37],17p [35,36], 18q [32,35,36], and 22q [36]; losses ofchromosomes 8p (59%), 1p (56%), and 18p (48%);and gains/amplification of chromosomes 13q (48%), 7p(44%), 8q (44%), and 20q (85%)—including amplifi-cation of 20q13 (38%) [32,35]. In agreement with theseobservations, all of our metastatic tumours showedcomplex karyotypes with seven or more altered chro-mosomes; at the same time, additional abnormali-ties were also observed in metastatic tumours fora large number of the above chromosomes (chro-mosomes 3, 7, 8, 9, 13, 17, and 20 in the wholeseries, and chromosomes X and Y in males). Inter-estingly, two different types of additional chromo-some abnormalities were found in metastatic versus

primary tumours: (1) abnormalities consisting of fur-ther gains/amplification of the 8q24, 11q, 13q, and20q chromosomal regions (in cases where the pri-mary CRC tumour samples already carried alterationsof these chromosome regions) and, less frequently,(2) new abnormalities of chromosomes 14q32, 10q23,6, and 19q13.

At present, there is no consensus about the geneticprofiles of localized versus metastatic disease in col-orectal cancer. In fact, according to the oncogenicmodel for CRC, in the metastatic scenario the sequenceof acquisition of chromosomal abnormalities does notappear to apply to individual tumours unless othermechanisms are involved (eg gene mutation, epige-netic silencing). In fact, in these tumours, early geneticevents in the model are frequently translated by relatedchromosomal abnormalities that are only acquired atrelatively late stages of the disease in our tumours.As an example, in our series, six tumours showeddel(17p) in the absence of del(18q) in the primarytumour; in turn, del(18q) was detected in only oneof the metastatic tumours from these individuals, thischromosomal region showing a normal diploid profilein the metastatic tumours from the other five patients.In another tumour, del(17p) was detected in the livermetastasis but not in its paired primary tumour. Finally,three tumours showed neither del(17p) nor del(18q) ineither their primary tumours or paired metastases. Incontrast, although it has been suggested that inactiva-tion of the DCC tumour suppressor gene would occurduring relatively early stages of tumourigenesis, our



Figure 4. Genetic heterogeneity of primary colorectal carcinomas: hypothetical intratumoral aneuploidization pathways for the mostfrequently altered chromosomes (chromosomes 1, 7, 8, 11, 13, 17, 18, and 20). Percentages show the frequency of cases with a tumour cellclone displaying a specific cytogenetic profile. Genetic aberrations common to all tumour cell clones within individual tumor samples areshown in bold; - indicates chromosome deletion or loss and + indicates gain or amplification of specific chromosomes and chromosomeregions, respectively. A detailed description of each case (numbered outside the circles at the top of every pathway) and the list of genesassociated can be found in the Supporting information, Supplementary Table 2.

Figure 5. Representative pictures of cell nuclei from a patient carrying two different tumour cell clones in the primary tumour andjust one clone in their paired liver metastases, as defined by simultaneous hybridization for chromosomes 8p22 (red spots), 8p11(blue spots), and 8q24 (green spots): one clone shows one copy for 8p22, two copies for 8p11.1, and three copies for 8q24, whilethe other has acquired an extra copy for chromosome 8p11.1 (n = 3) and two additional copies (n = 5) for chromosome 8q24. Thelatter clone co-existed in the primary tumour and the metastatic sample, while the former could only be identified in the primarylesion.

own results suggest that chromosomal abnormalitiesassociated with deletion of one copy of this gene couldplay an important role in the metastatic stage, sincedel(18q) was found more frequently in liver metas-tases than in paired primary tumours. In keeping withthis, Paredes-Zaglul et al [12] pointed out the potentialassociation between acquisition of a metastatic phe-notype and loss of specific chromosomal regions inCRC, as reflected by the fact that genetic changes suchas losses involving 18q and 17p frequently accounted

for the differences between primary and metastatictumours. Also, Weber et al [38] provided evidencethat both chromosomal losses (in addition to lossesof chromosome 8p) are associated with the progres-sion of colorectal cancer and the occurrence of distantmetastases. Our results expand on these observationsby showing that gains/amplification of the 8q, 11q,13q, and 20q chromosomal regions probably reflect apro-metastatic karyotype due to their relatively higherfrequency in liver metastases. Interestingly, in most of



Table 4. Correlation between the number of signals obtained foreach individual iFISH probe in primary colorectal tumours (n = 24)versus their paired liver metastases (n = 24)

Chromosomal Chromosomalregion regionidentified by identified bythe iFISH probe R2/p value the iFISH probe R2/p value

Tel 1p 0.84/<0.001 11p11.1 0.77/<0.0011p36 0.84/<0.0011q25 0.60/0.002 11q22 0.67/<0.0012p11.1 0.91/<0.001 12p11.1 0.73/<0.0012p24 0.89/<0.001 12p13 0.68/<0.0013p11.1 0.73/<0.001 12q13 0.71/<0.0013q26 0.60/0.002 13q14 0.79/<0.0014p11.1 0.91/<0.001 13q34 0.59/0.0034p16.3 0.93/<0.001 14q32 0.80/<0.0015p15.2 0.77/<0.001 15p11.1 0.88/<0.0015q31 0.67/<0.001 15q22 0.83/<0.0016p11.1 0.74/<0.001 16q11.1 0.85/<0.0016q21 0.65/0.001 17p11.1 0.83/<0.0016q23 0.69/<0.0017p11.1 0.64/0.001 18p11.1 0.69/<0.0017q31 0.60/0.002 18q21 0.55/0.0068p11.1 0.77/<0.001 19q13 0.74/<0.0018q24 0.59/0.003 20p11.1 0.50/0.0138p22 0.69/<0.0019p11.1 0.83/<0.001 21q22 0.75/<0.0019p21 0.82/<0.001 22q11.2 0.78/<0.0019q34 0.65/0.001 Xfp11.1 0.92/<0.00110p11.1 0.62/0.001 Xmp11.1 0.85/<0.00110q23 0.60/0.002 Yq12 0.90/<0.001

11q13 0.40/0.05

17p13 0.43/0.04

20q13.2 .44/0.0340

R2 = coefficient of correlation; Xf = chromosome X in females; Xm =chromosome X in males; iFISH = interphase fluorescence in situ hybridization.Chromosome regions showing the lowest degree of correlation (p ≥ 0.01) arehighlighted in bold.

the tumours, these chromosomal regions were gainedthrough aneuploidy in a clone which had undergonetetraploidization (see Supporting information, Supple-mentary Table 2), supporting the notion that aneuploidgains are critical for the progression of invasive carci-nomas to metastatic tumours. In this regard, it has beenpreviously shown that in such cases, chromosome 20qcan be either tetraploid/polyploidy or aneuploid andthat tetraploidy could be a status itself rather than atransient stage [39].

Among our tumours, chromosome 8 was the mostfrequently altered individual chromosome. Its abnor-malities consisted of whole chromosome gains (asconfirmed by SNP-array; Sayagues et al, unpublishedobservations), del(8p22), and/or gains/amplification of8q24, whose frequency slightly increased from primaryto metastatic tumours. Interestingly, the specific intra-tumoural cytogenetic profiles observed for this chromo-some suggest that numerical abnormalities of the entirechromosome 8 may precede the occurrence of struc-tural abnormalities specifically involving the 8q and 8pchromosomal regions. Of note, structural abnormalitiesof chromosome 8 appeared to be independent of otherchromosomal abnormalities, since they did not showany statistically significant association with abnormal-ities of other chromosome regions. Previous studieshave reported that overexpression of the PRL-3 gene,

Table 5. Numerical changes detected by iFISH in liver metastases(n = 24) versus paired primary tumours (n = 24) for the 8q24,11q13, 13q34, 17p13, and 20q13 chromosome regions.Chromosomal changes between both groups are highlighted inbold

No of copies detected by iFISH(Primary tumour/liver metastases)

Case 8q24 11q13 13q34 17p13 20q13

1 4/4 4/4 7/8 1/3 6/62 4/4 4/4 8/9 2/2 6/63 4/5 4/4 5/4 2/2 5/44 6/5 2/2 2/2 1/1 4/45 3/4 2/3 3/5 1/3 4/66 3/3 2/3 4/4 1/1 4/47 3/3 3/3 2/1 1/1 2/28 6/6 2/2 2/2 1/1 3/39 5/5 3/3 4/5 2/2 6/6

10 3/4 3/3 4/5 3/3 5/611 3/4 3/3 5/6 2/3 5/412 8/10 5/5 3/3 2/2 8/813 amp/amp 3/3 3/3 2/2 5/514 2/2 3/2 3/3 1/1 2/215 5/6 4/8 5/5 3/3 4/816 2/2 2/4 2/2 2/1 4/517 7/7 7/7 2/2 5/518 3/8

4/52/8 3/5 1/4 5/10

19 5/5 3/10 6/6 2/2 8/820 2/2 2/5 2/2 1/1 2/221 5/5 4/4 6/6 3/3 5/522 3/5 2/2 2/3 1/1 5/623 2/2 2/2 5/6 2/2 6/624 3/3 2/2 3/3 2/2 2/2

amp = amplified iFISH signal.

which maps to chromosome 8q24, is associated withthe metastatic process in CRC [40]. In addition, thischromosomal region has been shown to contain sin-gle nucleotide polymorphisms (SNPs) associated withan increased risk of development of CRC [41], whichwould support its role in the multi-step oncogenic pro-cess of this group of tumours. In addition to chro-mosome 8, the co-existence of gains/amplification inchromosomes 13q and 20q in the same tumour cellclone was also detected in the majority of tumours,most of which showed five or more copies of the gainedregions. Preferential amplification of 13q and 20qcould be particularly relevant to the metastatic poten-tial of CRC, since both chromosome regions have beeninvolved in the progression of adenomas to carcinomas[42,43] and metastatic tumours [32,44–46]. In line withthese findings, Xiao et al [47] found 20q13 gains in2/10 non-metastatic versus 11/14 metastatic tumours;similarly, Hidaka et al [45] found an increased num-ber of copies for the 20q13.2 region in CRC withliver metastasis (16/18 tumours) versus non-metastaticprimary CRC lesions (7/17). Additional chromoso-mal abnormalities frequently found in our study inboth primary tumours and their paired liver metastasesincluded del(1p), del(5q), and gains of chromosome7, and both nulisomy Y and gains of chromosome Xin males. Although all of these abnormalities have alsobeen reported in colorectal cancer tumours [48–50], the



exact significance of their high frequency in patientswith liver metastases remains to be elucidated.

Intratumoural pathways of clonal evolution inprimary versus metastatic CRC tumoursIn the present study, we applied multi-colour iFISH forthe analysis of the genetic diversity of primary colorec-tal tumours and their liver metastases in an attempt togain insight into the most frequent pathways of intra-tumoural clonal evolution in tumour cells carrying agenetic background of metastatic CRC. Interestingly,our results show a relatively high degree of intra-tumoural cytogenetic heterogeneity in primary CRCassociated with liver metastasis, since two or moredifferent cytogenetically related tumour cell cloneswere identified by multi-colour iFISH in all but onetumour. Although small changes with the acquisitionof additional chromosomal abnormalities were foundin tumour cell clones from liver metastases from nearlyall patients, a strong coincidence was observed betweenthe cytogenetically defined tumour cell clones presentin primary versus paired metastatic tumours. Takentogether, these results are in keeping with those pre-viously reported by other authors in relatively smallseries of patients (6–38 tumours) using conventionalcytogenetics, iFISH for a limited number of probes,CGH or SNP-arrays in which the overall chromoso-mal abnormalities found in primary versus metastatictumours were compared [12,28,37,44,51,52]. However,the techniques and approaches used in those studies didnot permit the identification of chromosomal abnor-malities restricted to relatively small subpopulationsof tumour cells present in the sample and/or triploid,tetraploid, and other multiploid karyotypes. To the bestof our knowledge, this is the first study in which thecytogenetic heterogeneity of colorectal tumours hasbeen analysed at the intratumoural cell level. Interest-ingly, in about three-quarters of all metastatic tumours,the predominant tumour cell clone was also highlyrepresented in the paired primary tumours, while bycontrast, in a lower proportion of cases, a minorclone present in the primary tumour became the dom-inant one in the liver metastases. Metastatic tumoursexhibited a preferential acquisition of chromosomalabnormalities involving the 11q13, 17p13, and 20q13regions in specific tumour cell clones, together with agreater frequency of new, but variable, abnormalitiesof chromosomes 14q32, 10q23, 6, 15q22, and 19q13.Together, these results suggest the presence of genesrelevant to the metastatic process in these chromosomeregions, particularly when they are associated witha cytogenetic background in which abnormalities of6q23, 9q34, 12p13, 13q, and 18q21 are already present,as supported by the significant correlation found inliver metastases between these and the former cyto-genetic abnormalities. In line with these findings, pre-vious studies in which primary CRCs were comparedwith liver metastases also reported a higher frequencyof abnormalities involving chromosomes 4, 6, 7, 8, 11q,

13q, 17, 18q, and 20q [24,28,32,38,44] in metastaticversus primary tumours, in addition to a preferentialloss of chromosome 3p [36]. Taken together, theseresults suggest that the overall cytogenetic backgroundof the primary tumour could determine the proba-bility of distant liver metastasis; the acquisition ofadditional abnormalities, in particular those of chro-mosomes 11q13, 17p13, and 20q13, would potentiallyfavour its occurrence. The specific requirement of indi-vidual abnormalities to the metastatic process or theadaptation to the micro-environment deserves furtherinvestigation [53,54].

In conclusion, our results confirm the existence ofa clear association in metastatic CRC between theoccurrence of liver metastases and complex karyotypes.Interestingly, despite the similarities of the differenttumour cell clones present in primary tumours and theirpaired metastases, additional chromosomal abnormal-ities were frequently observed, suggesting that suchalterations could be associated with the metastatic pro-gression of CRC.

AcknowledgmentThis work was partially supported by grants from theConsejeria de Sanidad, Junta de Castilla y Leon, Val-ladolid, Spain (SAN191/SA09/06 and SAN673/SA39/08); Fundacion Memoria de Don Samuel SolorzanoBarruso, Salamanca, Spain; Caja de Burgos (ObraSocial), Burgos, Spain; Grupo Excelencia de Castillay Leon (GR37); and the RTICC from the Instituto deSalud Carlos III (ISCIII), Ministerio de Sanidad y Con-sumo, Madrid, Spain (RD06/0020/0035-FEDER). JMSayagues and M Gonzalez were supported by grants(CP05/00321 and FI08/00721, respectively) from theISCIII, Ministerio de Ciencia e Innovacion, Madrid,Spain. We would like to thank Dr Manuel Medina andthe Red de Bancos de Tumores de Andalucıa (Granada,Spain) for contributing to this study.

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SUPPORTING INFORMATION ON THE INTERNET

The following supporting information may be found in the online version of this article.

Supplementary methods. Statistical studies and relevant clinical data about the 24 patients included in the present study.

Table S1. Frequency of distinct numerical and structural chromosomal abnormalities identified for each chromosome and chromosomeregion analysed in 24 primary sporadic colorectal tumours and their paired liver metastases.

Table S2. Metastatic colorectal cancer. Detailed description of each of the tumour cell clones present in primary tumours (n = 24) andtheir paired metastatic (n = 24) tumour samples and their frequency in said samples.

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