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Prospective Assessment of Allelic Losses at 4p14–16 in Colorectal Cancer: Two Mutational Patterns and a Locus Associated with Poorer Survival¹

Institut de Recerca Oncològica [R. A., R-A. R., L. M., S. T., G. A., M. A. P.] and Institut Català d’Oncologia [G. C.], Hospital Duran i Reynals, L’Hospitalet, 08907 Barcelona; Departament de Biologia Cellular i Fisiologia, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, 08913 Barcelona [M. R., R. M.]; and Servei d’Oncologia and Laboratori d’Investigació Gastrointestinal, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona [E. M., G. C.], Spain

	ABSTRACT

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Previous studies have shown that allelic losses in a locus mapping to the chromosomal region 4p14–16 are indicative of poor prognosis in colorectal cancer. To further characterize the region involved and to confirm earlier observations, we have analyzed losses of heterozygosity (LOH) in nine microsatellite markers spanning this region in a prospective series of 181 colorectal carcinomas. The extent and the nature of the allelic imbalance were also ascertained by comparative genomic hybridization analysis of selected cases. The minimum common deleted region was confined to marker D4S2397 (LOH in 35% of the informative cases). Surrounding markers displayed LOH in 13–25% of informative cases and (other than the D4S2397 marker itself) showed a higher rate of allelic imbalances in association with mutations in the p53 tumor suppressor gene. Tumors with lymph node invasion also displayed increased rates of LOH in most markers. Regarding patient outcome, LOH solely at the D4S2397 locus was indicative of a shorter disease-free survival (P = 0.027). In consequence, two patterns of allelic loss are defined within the 4p14–16 region: (a) gross losses associated with tumor progression and probably attributable to the genomic instability related to the inactivation of the p53 tumor suppressor gene; and (b) specific losses limited to the D4S2397 locus (within an estimated fragment of 2 Mb) and associated with increased tumor aggressiveness. The presence of one or more putative tumor suppressor genes in this region is postulated.

	INTRODUCTION

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Researchers have been progressively concerned in the analysis of chromosome losses because they are commonly linked to tumor suppressor genes in colorectal and other types of neoplasias (1) . Deletions involving relevant tumor suppressor genes are usually indicators of tumor aggressiveness; illustrative examples are losses at 17p (p53 gene) and 18q (where the DCC and other putative tumor suppressor genes map; Refs. 2, 3, 4 ). Identification of the genes that are the specific targets of the observed alterations is puzzling because of the diversity of molecular processes involved in tumor progression and the presence of different forms of genomic instability that "randomly" disrupt the genome structure (5) . In addition, microallelotyping may lead to ambiguous results, because it only detects allelic imbalances that may be a consequence of loss or gain (6) .

DNA fingerprinting by AP-PCR⁵ (7) of paired normal and tumor tissues represents a molecular alternative to cytogenetics of solid tumors (6 , 8) . The unbiased nature of this technique and its ability to differentiate between losses and gains (6 , 8 , 9) provides a picture of imbalances in multiple chromosomal loci and an estimation of the genomic damage sustained by the tumor cell (10) . Steady analysis by AP-PCR of a series of 55 colorectal tumors revealed the existence of several loci displaying recurrent imbalances associated with a poor patient outcome (11) . One of such loci mapped to chromosomal region 4p14–16. To better define the lost region in the short arm of chromosome 4, we analyzed LOH in nine microsatellites spanning 4p14–16 in an unrelated collection of colorectal tumors. To confirm these results, CGH analysis was also performed in a subset of samples. Two patterns of allelic loss with distinctive molecular and clinicopathological correlations were observed in the investigated region, suggesting the presence of complex mutational mechanisms and/or different selective pressures. The minimum common deleted region was centered at marker D4S2397 (4p14–15) and correlated with poor patient outcome.

	MATERIALS AND METHODS

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Patients.
In the period between July 1991 and December 1993, a total of 198 patients preoperatively diagnosed with colorectal cancer and operated upon in the Hospital de la Santa Creu i Sant Pau were prospectively included in a study designed to evaluate the clinical usefulness of genetic alterations. Inclusion criteria were: (a) electively operated primary adenocarcinomas; (b) the obtainment of fresh paired normal mucosa-tumor samples within 2 h after tumor removal; (c) no postoperative death; and (d) follow-up data available. Inclusion in this study did not influence the adjuvant treatment given, and the protocol was approved by the local Ethics Committee. No chemo- or radiotherapy was given prior to surgery. Cases were pathologically staged using the Astler-Coller modification of the Dukes’ classification system. Carcinomas and paired normal samples were collected as fresh specimens and snap frozen within 2 h after removal and then stored at -80°C. Follow-up status was updated every 6 months. The end point of the study was January 1999.

In eight cases, LOH analysis was not possible because of the low quality of DNA. Nine cases displayed ubiquitous microsatellite instability and were excluded. The remaining 181 patients have been the subject of the present analysis. Mean age was 67 ± 11 years (range, 33–96), and sex distribution was 105 males and 76 females. Dukes’ stage was as follows: 23 A+B1, 74 B2+B3, 49 C, and 35 D. One hundred and eight patients (60%) were N₀ (no lymph nodes with metastasis), 45 (25%) were N₁ (1–3 positive nodes), and 28 (15%) were N₂ or N₃ (four or more positive nodes/metastasis in a named vascular channel or apical node). Forty-four tumors (24%) were located in the right colon, and 137 (76%) were in the left colon. In 38 patients (21%), tumor resection was performed for palliative purposes, and disease persisted after surgery. In the other 42 cases, disease recurred during follow-up. Mean follow-up was 64 ± 17 months (range, 6–89). Average time of relapse was 22 months (range, 3–72). Seventy-four patients (36 without persistent disease and 38 with persistent disease) died of disease.

DNA Extraction.
Transformed cells constituted 50% or more of the tumor tissue specimen, as assessed by histological examination. Genomic DNA was extracted by the phenol/chloroform method. DNA was diluted to a concentration of 20 ng/µl, and 1 µl of each DNA was analyzed in a 0.75% agarose gel and stained with ethidium bromide to verify its quality and concentration.

Analysis of LOH.
Nine microsatellite markers (D4S2946, D4S1551, D4S2948, D4S3022, D4S2397, D4S418, D4S2912, D4S1587, and D4S405) mapping to 4p14–16 and spanning an estimated area of 7–8 Mb were analyzed for LOH. The order and physical genetic mapping (fig. 1) is based upon the Genetic Location Database (12) . The relative position of most markers coincided with that reported in other maps, including the Genethon human genetic linkage map (13) , the Unified Database for Human Genome Mapping,⁶ the Genome Database,⁷ and the Stanford maps.⁸ The PCRs were performed with 50 ng of genomic DNA, PCR reaction buffer (Boehringer Mannheim, Mannheim, Germany), 125 µM each deoxynucleotide triphosphate, 1 µM primer, 2.5 mM Mg₂Cl, 2 µCi [-³²P]dCTP (Amersham, Buckinghamshire, United Kingdom), and 1 unit of Taq DNA polymerase (Boehringer Mannheim) in a final volume of 25 µl. The reactions were optimized for each set of primers and consisted of an initial denaturation step (94°C for 45 s), 35 cycles (94°C for 45 s; 55–65°C for 45 s; and 72°C for 1 min), and a final extension (72°C for 5 min). PCR products were separated by electrophoresis in denaturing 6% polyacrylamide 8 M urea sequencing gels and exposed to X-ray film.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Deletion map in nine microsatellite markers located in 4p14–16. Markers are ordered from the telomere (up) to the centromere (down) according to the Genetic Location Database. The physical mapping (LDB) is shown in Mb. Illustrative examples of allelic patterns are depicted: unique LOH at D4S2397 (cases 39, 51, and 164), confined areas of loss (cases 65, 138, and 174), and extensive LOH (cases 57, 124, 157, 176, 177, and 217). *, cases also analyzed by CGH (see Fig. 3 ).

CGH.
To further confirm the pattern of allelic deletion in chromosome 4, 11 cases displaying different degrees of allelic imbalances at 4p were analyzed by CGH. Cases were selected based on the extension of the deletion region as determined by microallelotyping: five cases with LOH in most of the markers and six cases with single or interspersed imbalances. Briefly, normal and tumor DNAs were labeled with Spectrum Red-dUTP and Spectrum Green-dUTP by nick translation using a commercial kit (Vysis, Downers Grove, IL). Control experiments, in which the labels were interchanged between normal and tumor, were performed to confirm chromosomal alterations. Subsequently, equal amounts of the labeled probes (300–500 ng) and 10 µg of Cot.1 DNA were coprecipitated using ethanol. DNA was dissolved in 12 µl of hybridization buffer (50% formamide, 10% dextran sulfate, 2x SSC, and 1% Tween 20, pH 7.0) and denatured at 73°C for 5 min. Normal metaphase spreads were denatured in 70% formamide, 2x SSC for 5 min at 73°C, and hybridized with the DNA mixture in a moist chamber for 2–3 days. Slides were washed according to the protocol supplied by the manufacturer. Slides were analyzed using a Cytovision Ultra workstation (Applied Imaging, Sunderland, United Kingdom).

Molecular Analysis.
Mutations at codons 12 and 13 of the K-ras gene were determined in 156 cases. Mutations in exons 4 to 9 of the p53 gene were analyzed in 143 tumors. Methods and results have been described elsewhere (16) .

Statistical Analysis.
Contingency tables were analyzed by Fisher’s exact test or ² test. For the survival analysis, patients with persistence of the disease after surgery (n = 38) were excluded. Five cases with Dukes’ D colon cancer underwent curative resection of the primary tumor and the metastasis and were included in the study. Disease-free and overall survival distributions were calculated by the Kaplan-Meier method and analyzed using the Log rank test. Multivariate analyses were performed using the Cox proportional hazards model. Statistical analysis was performed with SPSS software. All Ps are estimated from two-sided statistical tests.

	RESULTS

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Losses at 4p14–16.
Frequencies of LOH at each assessed locus are shown in Table 1 . The D4S2397 marker displayed the highest rate of LOH (50 of 143 informative cases). Fig. 1 depicts the detailed mapping of allelic losses at 4p14–16 in selected cases. The minimum common deleted region was centered at the D4S2397 locus and was confined between markers D4S3022 and D4S418, defining an area of 2 Mb based on the Genetic Location Database (12) . LOH at the D4S2397 locus with retention of heterozygosity in the rest of the informative markers was observed in 23 tumors (16% of all informative cases; 47% of cases with LOH at D4S2397; see cases 39, 51, and 164 in Fig. 1 ). Cases with LOH at D4S2397 showed retention of the adjacent centromeric marker D4S418 in 58% of informative cases. The neighboring telomeric marker D4S3022 was retained in 73% of the cases. Examples of microsatellite analysis and the allelic imbalance scoring are shown in Fig. 2 .

View larger version (58K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Illustrative examples of the analysis of LOH in three tumors. Arrowhead, allelic imbalances between normal (N) and tumor (T) tissue. The microsatellite short name is shown below each band pattern.

View larger version (47K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. CGH analysis of six colorectal carcinomas (top, case number). Normal and tumor DNAs were labeled with Spectrum Red-dUTP and Spectrum Green-dUTP. Control experiments, in which the labels were interchanged between normal and tumor, were performed to confirm chromosomal alterations. Chromosome 4 ideogram and CGH profile are depicted next to an example for each case. The number of chromosomes analyzed (n) is indicated below the profile.

Survival Analysis.
For survival analysis, only patients undergoing radical surgery were considered (n = 143). LOH at D4S2397 were associated with a diminished disease-free (Log-rank P = 0.027; risk ratio, 2.1; 95% CI, 1.1–4.0) and overall survival (Log-rank P = 0.046; risk ratio, 2.0; 95% CI, 1.0–4.2). Mean disease-free survival time for patients with allelic imbalances at D4S2397 was 57 months (95% CI, 46–67) versus 72 months (95% CI, 66–79) for cases with no loss at this locus (Fig. 4) . When multivariate Cox analysis was performed in front of Dukes’ stage, LOH at D4S2397 still represented an increased risk (1.7; 95% CI, 0.9–3.5), although it did not reach statistical significance (P = 0.112). In consequence, it was not an independent prognostic factor. The allelic status of the rest of the markers analyzed and gross LOH did not appear to affect patient outcome (data not shown).

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Kaplan-Meier disease-free survival curves for the allelic status of marker D4S2397. Recurrence of the disease was higher in patients displaying LOH at this locus in front of the ones retaining both alleles (no LOH). Cases with persistent disease (n = 38) were excluded from the analysis.

	DISCUSSION

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Analysis by unbiased AP-PCR of a series of colorectal carcinomas provided allelic information (loss or gain) on 100 anonymous genomic markers (11) . Losses at several sequences in chromosome 4 were associated with a diminished survival, and the putative presence of a tumor suppressor gene in the proximity of one of these loci was hypothesized (11) . Furthermore, allelic gains (that might be misconceived as losses in microallelotyping studies) were uncommon in markers mapping to the short arm of chromosome 4 (11) . After a limited microallelotyping analysis, the region with a higher rate of losses was located to 4p14–16, where the AP-PCR-generated marker was located (11) . To confirm and to further characterize the chromosomal region affected, we performed a detailed analysis in a new series of 181 colorectal tumors prospectively collected. In agreement with our initial exploratory study, we show here that losses at one of the markers (the D4S2397 locus) within this region are indicators of poorer survival, reinforcing the hypothesis that this alteration affects tumor biology.

The hypothesis that a tumor suppressor gene lies near the D4S2397 marker is based in a sequence of observations: (a) the highest frequency of LOH in our series of colorectal carcinomas is in marker D4S2397 (Fig. 1) ; (b) losses at this locus are associated with more aggressive tumors (Fig. 4) ; and (c) more than one-half of the tumors displaying losses at this marker showed retention of the two flanking markers, which strongly suggests that this region is the one with a higher pressure to be lost in the tumorigenic process.

Further evidence supports the presence of a tumor suppressor gene in the short arm of chromosome 4. These include the reversion of the immortal phenotype by chromosome 4 transfer (22) and the frequent occurrence of losses in or near the 4p14–16 region in bladder (14) and head and neck squamous cell carcinomas (23) . Additionally, a complex rearrangement, t(4;9;22)(p14;q34;q11.2), has been documented in a patient with aggressive leukemia (24) . Finally, in a study using the AP-PCR molecular karyotyping approach, Malkhosyan et al. (6) have reported an increased rate of chromosome 4 losses in metastases when compared with primary colorectal carcinomas, suggesting the presence of a gene related to tumor progression.

A striking observation in our study is the complex pattern of loss and retention at the examined region. Losses at D4S2397 occur in 35% of all colorectal carcinomas and seem to affect tumor aggressiveness independently of the p53 gene status. In turn, flanking markers, with a lower rate of imbalances, display more frequent allelic losses in invasive tumors and correlate with mutations in the p53 gene (Table 1) , although they are not indicative of a shorter survival. It is of note that in the same series of tumors, mutations in the p53 gene were indicators of a poorer outcome (16) . The differential behavior of LOH at D4S2397 and the neighboring markers suggests that distinct mechanisms and/or selection pressures participate in the mutational events that affect this chromosomal region during the tumorigenic process. Gross LOHs will correspond to actual deletions of large genomic regions, if not the entire chromosome, as we have shown by CGH analysis. These and other alterations, including moderate allelic gains (9) and gene amplification (25 , 26) , could be a mere consequence of the increased genomic disruption that accompanies tumor progression and the escape of the cell from the p53 genomic integrity checkpoint (27) . Alternatively, LOH at D4S2397, with retention or not of the flanking region, could be an earlier event favored by selective pressures, as hinted by its association with increased tumor aggressiveness. The nature of the different mutational phenomena is unknown, but it can be hypothesized that its occurrence is probably an outcome of recombinational repair mechanisms (27) . Increased chromosome breakage because of DNA metabolism defects has been reported to enhance the rates of allelic recombination and LOH (28 , 29) . As an alternative hypothesis, the presence of more than one gene relevant to tumor biology within 4p14–16 can be postulated. Therefore, diverse selective pressures (including genetic, epigenetic, and microenvironment determinants, as well as the timing in the progression process) may favor different genetic alterations, resulting in complex patterns.

In summary, we have used molecular karyotyping techniques (AP-PCR and CGH) concerted with microallelotyping to investigate the involvement of allelic losses at 4p14–16 in two independent series of colorectal carcinomas (Ref. 11 and this report). Losses at 4p14–16 display two patterns of clinicopathological correlates: (a) extensive LOHs are associated with p53 mutations and invasion; and (b) LOH affecting one of the markers (D4S2397) associates with increased tumor aggressiveness. These results support the hypothesis that one or more putative tumor suppressor genes lie near locus D4S2397.

	ACKNOWLEDGMENTS

 
We thank Victor Moreno for help with the statistical analysis.

	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 This work was supported by grants from Comisión Interministerial de Ciencia y Tecnología and Fundació La Marató de TV3. R. A. and S. T. were fellows of the Spanish Ministry of Education. R-A. R. is a fellow of Comissió Interdepartamental de Recerca i Innovació Tecnológica. L. M. is a fellow of the Asociación Española contra el Cáncer.

² Present address: Deutsches Krebsforschingszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.

³ Present address: Fondation pour Recherches Mèdicales, 64 Avenue de la Roseraie, CH-1211 Genève 4, Switzerland.

⁴ To whom requests for reprints should be addressed, at Institut de Recerca Oncològica, Hospital Duran i Reynals, Autovía Castelldefels km 2.7, L’Hospitalet, 08907 Barcelona, Spain. Phone: 34-93-2607775; Fax: 34-93-2607776; E-mail: mpeinado{at}iro.es

⁵ The abbreviations used are: AP-PCR, arbitrarily primed PCR; LOH, loss of heterozygosity; CGH, comparative genomic hybridization; CI, confidence interval.

⁶ Internet address: http://bioinformatics.weizmann.ac.il/udb/.

⁷ Internet address: http://gdbwww.gdb.org/gdb/.

⁸ Internet address: http://shgc-www.stanford.edu/Mapping/phys_map/Chr4YAC.html.

Received 5/11/99; revised 8/10/99; accepted 8/11/99.

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