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[Cancer Research 63, 5731-5737, September 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Common Genetic Evolutionary Pathways in Familial Adenomatous Polyposis Tumors1

Gemma Tarafa2, Esther Prat2, Rosa-Ana Risques, Sara González, Jordi Camps, Mónica Grau, Elisabeth Guinó, Víctor Moreno, Manel Esteller, James G. Herman, Josep-Ramon Germà, Rosa Miró, Miguel Angel Peinado and Gabriel Capellá3

Laboratori de Recerca Translacional [G. T., S. G., M. G., J-R. G., G. C.] and Servei d’Epidemiologia del Càncer [E. G., V. M.], Institut Català d’Oncologia, Barcelona 08907, Spain; Departament de Biologia Cellular, Fisiologia i Inmunologia, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain [E. P., J. C., R. M.]; Cancer Epigenetics Laboratory, Molecular Pathology Program, Centro Nacional de Investigaciones Oncológicas, Majadahonda 28220, Spain [M. E.]; The Johns Hopkins Oncology Center, Baltimore, Maryland 21231 [M. E., J. G. H.]; and Departament d’Oncologia Molecular, Institut de Recerca Oncològica, Barcelona 08907 [R-A. R., M. A. P.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cancer cells progress through the accumulation of genetic alterations. Familial adenomatous polyposis (FAP) tumors provide an excellent model to unravel the molecular steps underlying malignant transformation. Global genomic damage was assessed in 56 adenomas and 3 carcinomas from six FAP patients and compared with that of sporadic adenomas and carcinomas. Evolutive trees were traced after application of maximum likelihood clustering and split decomposition methods to the analysis of comprehensive genetic profiles generated by diverse molecular approaches: arbitrarily primed PCR, comparative genomic hybridization, and flow cytometry. Overall, genomic damage as assessed by arbitrarily primed PCR was lower in familial adenomas than in sporadic adenomas and carcinomas. Comparative genomic hybridization data also show a low number of alterations in the majority of FAP adenomas. Tumors of the same patient were likely to share specific genetic alterations and may be grouped into one or two clusters. Putative common pathways were also identified, which included tumors of up to three different patients. According to our data, FAP tumors accumulate specific genetic alterations and in a preferred order that is characteristic of each individual. Moreover, the particular genetic background and environmental conditions of a FAP patient restrain the molecular evolution portrait of synchronous tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The complexity and heterogeneity of the molecular processes driving cell transformation preclude the outline of progression models beyond theoretical postulates of limited practical application. Hereditary nonpolyposis colorectal cancers may be an exception, displaying very specific clinicopathological and molecular profiles, including clinical outcome. In this case, the evolutive driving force is the impairment of the DNA mismatch repair machinery, resulting in a specific mutator phenotype denoted by ubiquitous microsatellite instability (1) . FAP4 is a hereditary syndrome characterized by the presence of hundreds to thousands of adenomatous lesions in the colon at early ages that eventually transform into malignant adenocarcinomas. It is well known that the predisposition lays, in the majority of cases, in the presence of a defective APC gene (2) . Recently, alterations of the MutY homolog (MYH) have been associated with some classical FAP cases after a recessive pattern (3) . However, the genetic steps underlying the malignant transformation have been only partially elucidated.

Chromosomal instability has been blamed as the driving force in this type of tumor, but unlike in cancers with microsatellite instability, no characteristic fingerprints are observed (4) . In addition to APC, the genetic background and environmental factors may condition the evolutive pathway of these tumors. In fact, a recent study has demonstrated that the same defective APC gene in two strains of mice results in very different penetrance of the disease (5) , depicting the importance of modifying factors.

The study of cancer genomic disruption profiles may provide with clues to unveil the driving forces of tumor progression in FAP patients. Because of the heterogeneous nature of the alterations observed, comprehensive approaches are required to fully characterize the molecular patterns. We have used AP-PCR, which provides a comprehensive and unbiased estimation of the genomic damage (6) , jointly with CGH and flow cytometry. The molecular profiles and the type and degree of genetic damage of 56 adenomas and 3 carcinomas from six FAP patients have been assessed. Specific genetic alterations in the APC gene and neighboring regions have also been investigated. Molecular data have been analyzed with different statistical methods, including phylogenetic analyses. These approaches can shed light into the relationships between distinct adenomas, and they can be used to identify characteristic pathways of genetic evolution.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Material
Fifty-six adenomas and 3 colorectal carcinomas from five classical FAP cases (defined by the presence of more than 100 colonic adenomas) and one attenuated FAP were included. Six to 12 adenomas per case were analyzed. Main characteristics of FAP patients and tumors are shown in Table 1Citation . Age at diagnosis was high because most patients were not following screening programs, and five of six cases were de novo. In all cases, microscopically normal mucosa was available and analyzed concomitantly. Adenomas were collected from well-separated regions including distal, transverse, and right colon, although the precise location of each collected adenoma was not recorded. Cases were coded with letters, and adenomas were numbered consecutively after the letter code for a given case. "T" was used for carcinomas. In addition, for AP-PCR analysis, 10 paired normal mucosa-adenoma-sporadic carcinoma samples and 113 sporadic paired normal tumors-colorectal samples were studied. In all cases, tumor cell enrichment ensuring that 80% of all nuclei analyzed were tumoral was performed by manual microdissection. DNA was extracted from frozen sections by the phenol/chloroform method.


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  		Table 1 Clinical and molecular characteristics of FAP patients and tumors

 
Methods
APC Gene Alterations
Mutational Analysis.
Mutations at exons 1–12, 14, and the first 5.260 bp of exon 15 of the APC gene, covering 90% of the coding region and 95% of reported mutations in classical, attenuated, and late onset FAP, were analyzed by single-strand conformational polymorphism using fluorescein-labeled primers described previously (7) and, more recently, direct sequencing of the PCR products. In our experience, we have detected mutations, mainly small deletions, in 43 of 92 Spanish classical FAP families analyzed (47%). No attempt has been made to detect mutations at the 3' end of the gene or gross deletions. Samples were processed in the ALFexpress DNA Sequencer and analyzed with Fragment Manager software (Pharmacia Biotech). Whenever an abnormal single-strand conformational polymorphism pattern was evidenced, sequencing was performed.

LOH and Haplotype of the APC Locus.
Two intragenic polymorphisms (nt 1458, RsaI; SspI at the 3' untranslated region) and two neighboring microsatellite markers (D5S299, D5S318) were used to perform haplotype analyses and, when comparing tumor and normal tissues, to analyze LOH at the APC locus. PCR products were labeled with {alpha}32P-dCTP and electrophoresed on 6% acrylamide/7 M urea sequencing gels.

Methylation-specific PCR.
DNA methylation patterns in the CpG islands of the APC gene promoters 1 and 1B spanning 7 and 6 CpGs, respectively, were determined as described (8) . Placental DNA treated in vitro with Sss I methyltransferase was used as a positive control for methylated alleles. DNA from normal lymphocytes was used as negative control.

Detection of Genetic Alterations by AP-PCR
The AP-PCR is a PCR-based DNA fingerprinting technique in which a single arbitrarily chosen primer is used to coamplify multiple and independent sequences by low-stringency conditions during the first cycles. Competition between these annealing events results in the reproducible and quantitative amplification of multiple bands. AP-PCR was used in our study because it produces fingerprints (of tagged bands) of moderate complexity that can be compared easily.

Primer Selection and Band Analysis.
Three different AP-PCR experimental conditions (with a single primer or sets of primers) were selected based on pattern reproducibility and readability (6 , 9) . The primers used were: 331DB2, 5'-ACAGATCTGAAGGGTGAAATATTCTCC-3'; Blue, 5'-CCGAATTCGCAAAGCTCTGA-3'; D12S77a, 5'-GAGGGCAACAACAGTGAA-3'; D12S77b, 5'-CTTTTTTTTCTCCCCCACTC-3'. Assay conditions were performed as described previously (for primers 331DB2 and D12S77, see Ref. 6 ; for primer Blue, see Ref. 9 ). All samples included were analyzed in duplicate. Films were scanned (Epson GT8500 with transparency unit). Intensity of bands was quantified using Phoretix 1D Advanced version 3.0 (Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom). Absolute values were corrected for background signal and interexperiment variability. A total of 122 different bands, informative for all cases, were analyzed.

Definition of Changes of Intensity.
To define a significant change in intensity when comparing tumor and normal tissues, intra-assay reproducibility was assessed. Four distinct AP-PCR reactions of a given paired tumor-normal DNA sample were performed in quadruplicate. The mean and SD of the intensity of the bands were calculated. A clear relationship was observed between band intensity and SD, more intense bands showing the highest SD. Therefore, a change of intensity was considered significant when the difference was above the 95% confidence interval in regard to a regression constant, depending on band intensity and calculated in reproducibility experiments (data not shown). Alteration status of every AP-PCR-informative band in the normal-tumor paired tissues was scored as 0 (no difference), +1 (gain in tumor), and -1 (loss in tumor).

Quantitation of Genomic Damage.
An index estimating the overall degree of genomic damage was calculated. The gains fraction is the sum of positive changes (gain in tumor) divided by the total number of informative bands, and the loss fraction is the sum of negative changes (loss in tumor) divided by the total number of informative bands (7) . Finally, the GDF was calculated as the sum of gains fraction and loss fraction.

Chromosomal Localization.
Chromosome assignment and localization of given bands was performed following procedures described previously (9) . For chromosome assignment, the monochromosome human/rodent hybrid (National Institutes of General Medical Sciences mapping panel 2; Coriell Institute for Medical Research, Camden, NJ) was used. A more refined subchromosomal assignment was achieved after using the Standford G3 Radiation hybrid panel (Research Genetics, Inc., Huntville, AL).

CGH
The technique used has been described previously (9) . Slides were analyzed using Cytovision Ultra workstation (Applied Imaging, Sunderland, United Kingdom) and Quips Smart Capture Software from Vysis (Downers Grove, IL). Ratio values above 1.20 and below 0.80 were considered to represent chromosomal gains and losses, respectively. A high-level DNA amplification was considered when the fluorescence ratio values exceeded 1.5 and, in addition, a distinct band-like hybridization signal of the tumor DNA was seen. Negative control experiments were performed using differentially labeled male versus male DNA and female versus female DNA. In addition, control experiments in which the Red-dUTP and Green-dUTP labels were interchanged between normal and tumor were also performed. CGH analyses were performed blindly regarding case ascription, and tumors of the same patient were not analyzed consecutively. Chromosomal regions 1pter, 16p, and 19 were excluded from the analyses.

DNA Flow Cytometry
Flow cytometry was performed from OCT-embedded fresh frozen tissue as described previously (10) . Normal mucosa was used as external control, showing a mean coefficient of variation (of the diploid G0-G1 peaks) of 3.43 ± 0.39. Aneuploidy was considered when the DNA content was above 1.1 (hyperdiploidy) or below 0.9 (hypodiploidy). Tumors with a DNA content between 1.1 and 1.3 were classified as near-diploid.

Statistical Analysis
All values are exposed as mean ± SD. Statistical differences between variables (adenoma versus carcinoma) were analyzed with unpaired t test or ANOVA, as appropriate. Either AP-PCR or DNA copy number as assessed by CGH were scored as 0 (no change), +1 (gains), or -1 (losses). Distance matrix was used to study similarity between every two adenomas. Values near 1 reveal a close relationship. Factor analyses were used to study similarity when all adenomas were considered. Both analyses were performed with SPSS software. Phylogenetic trees were assessed using continuous character maximum likehood clustering methods from the PHYLIP software (PHYLogenic Inference Package) version 3.5c (provided by J. Felsenstein, Department of Genetics, University of Washington, Seattle, WA).5 Tree drawing software (Treeview, version 1.6.1) was used to graphically display PHYLIP-generated data. Real evolutionary data often contain a number of different and, sometimes, conflicting signals that do not always support a unique tree. Therefore, a split-decomposition method implemented by Splitstree software version 26 was also used. In this case, less ideal data are not forced into a tree. Therefore, the software gives rise to a tree-like network that can be interpreted as possible evidence for different and conflicting phylogenies. Finally, changes of intensity in paired tumor-normal samples were analyzed with the ScanAnalyze/TreeView utilities (10) . Whenever these programs clustered adenomas in different categories, a general logarithm method was used to define those bands characteristic of each group (those showing Ps higher than 1.96).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
APC Gene Characterization of Patients and Tumors
We have detected an APC germline mutation in one of the six patients studied (Table 1)Citation . LOH at the APC locus was only detected in 1 of the 56 adenomas and in 1 of the carcinomas, both arising in the same patient. No promoter hypermethylation was detected. No mutation in the mutation cluster region of the APC gene was detected in the three carcinomas analyzed.

Chromosomal and Subchromosomal Alterations: Number and Profiles
The average GDF of the 56 FAP adenomas, as determined by AP-PCR fingerprinting, was 0.095 ± 0.02 (range, 0.073–0.1147; Table 1Citation ). There were no differences between adenomas when grouped by patient. These values were lower than that observed in sporadic adenomas (GDF, 0.131 ± 0.02; P = 0.05; Fig. 1ACitation ). This difference was more striking when adenomas were compared with carcinomas (GDF, 0.18 ± 0.11; P = 0.05; Fig. 1BCitation ).



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  		Fig. 1. Quantitation of genomic damage, as assessed by AP-PCR, in adenomas and carcinomas. A, comparison of FAP versus sporadic adenomas (AD). B, comparison of GDF of carcinomas versus all AD.

 
Both in adenomas and carcinomas, gains and losses were found at similar rates. Chromosomal assignment was possible in 49% of the 122 AP-PCR bands, being 70% of the chromosomes represented (1–12, 14, 17, 18, X). In adenomas, a clear trend to loss of chromosomes X and 3 was observed.

CGH was performed in 47 (45 adenomas and 2 carcinomas) of 59 samples. There were, on average, 2.5 alterations per adenoma (range, 0–20): 1.8 gains and 0.6 losses (Table 1)Citation . The majority of adenomas showed a low number of alterations (range, 1–5), and in 12 cases, no imbalances were detected (Table 2)Citation . The two FAP carcinomas analyzed by CGH (AT and ET) showed two and six chromosome abnormalities, respectively. The most frequent chromosome imbalances were gains of chromosome Xp (9 of 47 tumors), 9q34 (9 of 47 tumors), and 22 (6 of 47 tumors; Table 2Citation ). The more prevalent losses involved the whole chromosome X (12 of 47 tumors), whereas gain of this chromosome was detected in six tumors. Recurrent gains on 4p16, 10q, and 12q as well as losses on Xq and 18q were also present in two or more adenomas of distinct cases (Table 2)Citation .


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  		Table 2 CGH karyotype of FAP tumors

 
Relationships between Adenomas
Adenomas Display Common Genetic Features That Are Distinctive of Every FAP Patient.
CGH showed a strong genetic relationship among adenomas and carcinomas arising in a given FAP patient. In all cases, recurrent CGH changes can be detected in at least three adenomas (Fig. 2)Citation . In line with this observation, DNA content analysis also showed that adenomas arising in the same patient displayed the same diploid or hypodiploid (DNA content, <0.9) pattern (Table 1)Citation . However, factorial analyses of raw data of AP-PCR fingerprints did not provide relevant data. Several bands showed distinct patterns in different individuals. Because of the polymorphic display of these bands, tumors and paired mucosa of a given patient always clustered together.



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  		Fig. 2. CGH profiles showing recurrent chromosome imbalances occurring in distinct tumors of the same patient. A and B, similar gains on 2pterq22 and 3q21q23 were found in synchronous adenomas of case F (F1 and F3). C, identical losses on 7p21 were shared by the carcinoma (ET) and one adenoma (E1).

 
Genetic Heterogeneity in Adenomas of the Same FAP Patient.
The relative weight of polymorphisms in raw AP-PCR data precluded the identification of common genetic profiles among adenomas of distinct patients. Thus, data were normalized to reflect changes in intensity between normal and tumor tissue to allow for interpatient comparison (see "Statistical Analysis" in "Material and Methods"). Tree software packages showed that in four of the six cases (A, B, E, and F) at least two distinct subfamilies of tumors could be depicted (Fig. 3)Citation . Fingerprintings of the three carcinomas (AT, BT, and ET) were more complex but always related to one of the subfamilies of the adenomas. Interestingly, in the majority of the cases, a common putative ancestor regarding allelic losses or gains pattern could be envisioned for each subfamily (Fig. 3)Citation . Subfamilies of adenomas could not be evidenced in two cases (C and D).



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  		Fig. 3. Phylogenetic trees, using split decomposition analysis, of all FAP cases studied. AP-PCR trees are depicted. Adenomas are shown as identification letter of the FAP case, followed by numbers. N, normal mucosa; T, carcinoma.

 
This intracase heterogeneity was also observed when CGH data were analyzed. Despite the clear genetic relationship observed in tumors arising in a single patient, divergent evolution was also observed. In four of the six cases (Table 1)Citation , most of the adenomas could be tracked to a single pathway (data not shown). We conclude that significant genetic heterogeneity regarding specific allelic or chromosomal imbalances is present in synchronous FAP adenomas.

Phylogenetic Assessment of All FAP Tumors (Adenomas and Carcinomas) Arising in Distinct Patient Cases Shows Common Genetic Evolutionary Pathways.
Next, we analyzed whether this intracase heterogeneity was maintained among different FAP patients. Phylogenetic assessment of the AP-PCR fingerprints of all 56 FAP adenomas and 3 carcinomas was performed. Maximum likehood analysis revealed four main branches or putative pathways (Fig. 4Citation ; Ref. 11 ). In addition, a fifth one, containing only two adenomas from case B, was also observed.



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  		Fig. 4. A, Phylogenetic tree of all FAP tumors analyzed. {bullet}, adenomas (n = 56); {blacksquare}, carcinomas (n = 3); N, normal mucosa. Colors identify each FAP case: red, case A; violet, case B; deep red, case C; blue, case D; green, case E; yellow, case F. {blacksquare}{blacksquare}{blacksquare}{blacksquare}{blacksquare}{blacksquare}, lines divide tumors in the four main pathways identified; ·········, line depicts a putative fifth pathway including only two tumors (see "Results"). B, cluster analysis of AP-PCR data obtained from FAP adenomas and carcinomas. B1, ordering of samples (adenomas and carcinomas) has been forced to maintain the arrangement observed in Fig. 3Citation . Red depicts increases of intensity in tumors, whereas green depicts losses. B2, the common bands (in italics) and gain and loss ratio characteristics of the four pathways identified.

 
It is noteworthy that several changes occurred when phylogenetic analysis was performed with all adenomas when compared with assessment of individual cases. First, adenomas belonging to cases A [APC(+)] and B that showed distinct subfamilies, when considered individually, are now ascribed to a single pathway and in case B in conjunction with other adenomas. Second, case C adenomas, previously classified as not having distinct subfamilies, are now grouped into two distinct pathways, III and IV, shared with adenomas of other patients. Third, up to three of the four categories include adenomas of two or even three distinct FAP patients. Finally, in three patients (A, D, and F), all tumors were arranged in a single primary group. In the other three patients (B, C, and E), the tumors were grouped into two different primary groups. Altogether, phylogenetic analyses of all FAP adenomas and carcinomas strongly suggest common genetic evolutionary pathways be shared by three of the six FAP patients analyzed. This observation is even more striking, taking into account the strong similarity observed in adenomas arising in the same patient as assessed by matricial and factorial analysis. Unfortunately, the low number of alterations identified by CGH precluded this type of analysis using CGH data.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tumorigenesis is a multistep process involving the accumulation of somatic mutations and clonal expansions. The genetic dynamics of human tumor growth are still poorly understood, because serial observations are rare. Most colorectal tumors belong to the tumor suppressor pathway of which FAP tumors are among the clearest exponent (12) . FAP patients provide a natural model for the study of the role of genetic instability in early tumor growth and may be of help in gaining insight into the clonal dynamics in macroscopically independent tumors. We have used different techniques (AP-PCR, CGH, and flow cytometry) to study genetic instability in these tumors.

Here, we show that allelic imbalances (gains or losses) are consistently present in early colorectal adenomas, although at a low frequency when compared with carcinomas. In some cases, they are also associated with mild DNA content abnormalities or scarce chromosomal changes. In agreement with previous observations, a significant increase in the total number of allelic imbalances or chromosome alterations was observed in carcinomas, probably depicting the increased genetic instability observed in the adenoma-carcinoma transition. An APC gene mutation is believed to be necessary and sufficient to promote early adenoma growth (13 , 14) . Accordingly, we have detected no promoter hypermethylation and only occasionally LOH at the APC locus in the only APC(+) case that we have studied. This result suggests that the genetic abnormalities detected by AP-PCR and CGH, in this case, is likely to be secondary to APC gene inactivation. Recently, APC has been associated with the generation of chromosome instability (15) , although this issue remains controversial (16) . In the remaining cases with no APC alterations, it has not been elucidated yet their molecular basis that could be other APC gene alterations not detectable by direct sequencing or MYH glycosylase alterations (3) . In any case, the molecular nature of the first genetic alteration apparently has a profound impact on subsequent tumorigenesis (17) , because GDF of FAP adenomas are lower than that of sporadic ones.

Despite significant heterogeneity, synchronous adenomas of a given FAP patient showed common patterns of molecular evolution. In fact, phylogenetic assessment of the unbiased AP-PCR data evidenced multiple common nonrandom alterations among these complex networks. The lack of serial arrangement in most cases disregards a preferred order in the accumulation of genomic damage, although it is possible to track subfamilies of adenomas in a patient. These observations suggest that most genomic damage detected by AP-PCR is likely to be a manifestation of intrinsic genetic instability. In consequence, common genetic profiles might be more related to the susceptibility of certain chromosome regions to be altered rather than to selective pressures.

The strong relationship among synchronous adenomas was further confirmed by CGH analyses. In our set of tumors, a scarce number of chromosomal imbalances, as assessed by CGH, was observed. In a previous study (16) no imbalances were detected in a small series of FAP tumors with two APC mutational hits. Here, recurrent alterations were detected in synchronous tumors of the same patient, suggesting the existence of evolutive pathways in which a preferential order in the occurrence of chromosomal losses and gains was observed. This is in line with previous classical cytogenetic works in synchronous colorectal lesions (18 , 19) or CGH analyses in papillary renal cell carcinomas (20) . In both cases, a strong karyotypic similarity has been observed in independent synchronous lesions arising in the same (18 , 19) or different (20) patients. Thus, the genetic predisposition of FAP patients might condition the acquisition of genomic alterations in specific chromosomal subregions. Alternatively, selection of cells harboring specific chromosomal imbalances may have already occurred.

Arrangement of genetic profiles of independent tumors in phylogenetic trees may contribute to describe their historical relationships. FAP adenomas and carcinomas of different patients show common genetic evolutionary pathways. Concurrence of tumors from up to three patients was observed in three of the main branches. In addition, adenomas arising in the same patient may be ascribed to two different pathways. We hypothesize that restricted pathways are selected during early tumor progression in FAP patients. Therefore, a scenario in which hereditary-increased genetic instability continuously modulates clonal divergence could be postulated. Several factors may shape the molecular evolution of these tumors: (a) the genetic background (i.e., the molecular nature of APC gene mutation or other genetic alterations leading to overlapping phenotypes; Refs. 3 and 17 ) of the initiated clone; (b) a common embryonic origin; and (c) a shared exposure to carcinogens. Novelli et al. (21) have convincingly shown that up to 67% of microadenomas in a FAP patient may be polyclonal, requiring the interaction of multiple cells. This situation makes it further unlikely that multiple interaction results in common pathways unless restricted pathways are present.

Altogether, we have shown that allelic gains and losses occur early in FAP colorectal tumorigenesis, further supporting the multistep nature of tumorigenesis and perhaps related to the principal role of chromosomal instability in human colorectal tumorigenesis. Scarce chromosomal alterations are also present in a subset of adenomas. Phylogenetic assessment of these changes has shown that restricted nonrandom genetic pathways are allowed in the early stages of the disease, probably conditioning its biological behavior. These restricted pathways can only be seen in early growth. Once the carcinoma is established, the significant increase in genetic instability results in random proliferation (22 , 23) . The resulting extensive genomic damage precludes the identification of early restricted patterns that, nevertheless, have significantly influenced tumor biology.


   ACKNOWLEDGMENTS
 
We thank Ramón Seminago (Serveis Científico-Tècnics de la Universitat de Barcelona, Spain) for excellent technical support.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from Comisión Interministerial de Ciencia y Tecnología, Fondo de Investigación Sanitaria, and Fundació La Marató de TV3. G. T. is a fellow of the Ministerio de Ciencia y Tecnología. E. P. is a fellow of Institut Municipal d’Investigacions Mèdiques. R-A. R. is a fellow of Comissió Interdepartamental de Recerca i Innovació Tecnològica. J. C. is a fellow of Universitat Autònoma de Barcelona. Back

2 G. T. and E. P. contributed equally to this study. Back

3 To whom requests for reprints should be addressed, at Laboratori de Recerca Translacional, Institut Català d’Oncologia, Av. Gran Via s/n, km 2,7 08907 L’Hospitalet, Barcelona, Spain. Phone: 34-93-260-7952; Fax: 34-93-260-7466; E-mail: gcapella{at}ico.scs.es Back

4 The abbreviations used are: FAP, familial adenomatous polyposis; AP-PCR, arbitrarily primed PCR; CGH, comparative genomic hybridization; LOH, loss of heterozygosity; APC, adenomatous polyposis coli; GDF, genomic damage fraction. Back

5 Internet address: http://taxonomy.zoology.gla.ac.uk/rod/rod.html. Back

6 Internet address: ftp://ftp.uni-bielefeld.de/pub/math/plits/splitstree2. Back

Received 6/24/02. Revised 6/16/03. Accepted 7/ 8/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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L. Masramon, E. Vendrell, G. Tarafa, G. Capella, R. Miro, M. Ribas, and M. A. Peinado
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