High frequency allelic loss on chromosome 17p13.3–p11.1 in esophageal squamous cell carcinomas from a high incidence area in northern China

Jing Huang1,*, Nan Hu2,*, Alisa M. Goldstein2, Michael R. Emmert-Buck1, Ze-Zhong Tang2, Mark J. Roth2, Quan-Hong Wang2, Sanford M. Dawsey2, Xiao-You Han2, Ti Ding2, Guang Li2, Carol Giffen3 and Philip R. Taylor1,4

Cancer Institute and Hospital, Chinese Academy of Medical Sciences, Beijing 100021, People's Republic of China,
1 National Cancer Institute, NIH, 6006 Executive Boulevard, Room 321, Bethesda, MD 20892-7058, USA,
2 Shanxi Cancer Hospital, Taiyuan, Shanxi 030013, People's Republic of China, and
3 Information Management Service Inc., Silver Spring, MD 20904, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Allelic loss on chromosome 17p has been reported frequently in esophageal squamous cell carcinoma (ESCC) and generally encompasses the p53 locus at 17p13.1. However, a good correlation between allelic loss on 17p and mutation of p53 has not been found. This suggests the possibility that unknown tumor suppressor genes near p53 may be involved in the development of ESCC. To evaluate this possibility, we analyzed 30 microsatellite markers covering the entire short arm of chromosome 17 in 56 ESCC patients from a high risk population in northern China, including 34 with a family history of upper gastrointestinal (UGI) cancer and 22 without a family history of any cancer. Cancer lifestyle risk factors and clinical/pathological characteristics were also collected. We found frequent allelic loss (>=65%) at 28 of the 30 markers evaluated in these ESCC patients. The highest frequencies of allelic loss (>=80%) were found in three smaller regions: deletion region I located at 17p13.3–p13.2 (between D17S849 and D17S1828); deletion region II located at 17p13.2–p13.1 (between D13S938 and TP53); deletion region III located at 17p13.1–p12 (between D17S804 and D17S799). A number of genes have already been identified in these deleted regions, including: OVCA1, OVCA2 and HIC-1 in deletion region I; p53 in deletion region II; ZNF18, ZNF29, ALDH3 and ALDH10 in deletion region III. These results will help us direct future testing of candidate genes and narrow the search region for major new tumor suppressor genes that may play a role in the pathogenesis of ESCC.

Abbreviations: ESCC, esophageal squamous cell carcinoma; LCM, laser capture microdissection; LOH, loss of heterozygosity; UGI, upper gastrointestinal.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Esophageal cancer, with a 5 year survival below 10%, is one of the most common fatal cancers world wide. There is great geographical variation in the occurrence of this tumor, including exceptionally high risk areas such as Shanxi Province, a region in north central China with some of the highest esophageal cancer rates in the world (14). Epidemiological studies indicate that tobacco and alcohol are the major risk factors for esophageal cancer in the low risk populations of Europe and North America, but the etiology of this tumor in high risk populations remains less clear. Several studies in the high risk regions of China have demonstrated a strong tendency towards familial aggregation or tendency towards clustering of cases within families, suggesting that genetic susceptibility may play an important role in the etiology of esophageal cancer in these populations (59).

Previous molecular genetic studies have revealed multiple genetic alterations, including loss of tumor suppressor genes and activation of oncogenes, associated with the development of esophageal cancers (1017). Chromosomal regions with frequent allelic loss may point to major tumor suppressor genes that can assist our understanding of the molecular events involved in the development of esophageal cancer and may serve as the basis for development of markers for genetic susceptibility testing or screening for early detection of this tumor.

We have previously conducted two studies to better understand the genetic changes involved in the development of esophageal cancer in high risk Shanxi patients. The first study used a genome wide scan with 366 microsatellite markers in 11 esophageal squamous cell cancer (ESCC) patients with a family history of upper gastrointestinal (UGI) cancer and identified 46 markers, representing 14 regions, with very high frequencies (>=75%) of loss of heterozygosity (LOH) (18). In that study, 11 microsatellite markers on chromosome 17 were tested and one marker (D17S1303 at 17p13–p11) showed a very high frequency of LOH (80%) (18). Our second study used 18 of the 46 markers with a very high frequency of allelic loss, including D17S1303, to examine 46 ESCC patients, including 23 with and 23 without a family history of UGI cancer (19). In this study we also found frequent LOH at D17S1303 (in 100 and 89% of the two patient groups, respectively) (19).

Allelic loss on chromosome 17p has been reported frequently in esophageal cancer, including both ESCC and esophageal adenocarcinoma, and generally encompasses the p53 locus at 17p13.1 (16,2022). However, a correlation between LOH on 17p and p53 mutation has not been found. This suggests that unknown tumor suppressor genes near p53 may be involved in the development of esophageal cancer. To evaluate this possibility, we used 30 microsatellite markers covering the entire short arm of chromosome 17 to look for allelic loss in 56 ESCC patients from Shanxi province, China, that we have previously evaluated (18,19). In addition, we compared the LOH results with the cancer lifestyle risk factors for these patients and the clinical/pathological characteristics of their tumors to look for correlations.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection
Patients presenting in 1995 and 1996 to the Shanxi Cancer Hospital in Taiyuan, Shanxi Province, People's Republic of China, who were diagnosed with ESCC and considered candidates for curative surgical resection were identified and recruited to participate in this study. The study was approved by the Institutional Review Boards of the Shanxi Cancer Hospital and the US National Cancer Institute. For this study a total of 56 patients with ESCC were selected who had a histological diagnosis of esophageal squamous cell cancer confirmed by pathologists at both the Shanxi Cancer Hospital and the National Cancer Institute. None of the patients had prior therapy and Shanxi was the ancestral home for all. Of the 56 ESCC patients studied, 34 had a family history of UGI cancer (i.e. a first, second or third degree relative with cancer of the esophagus, gastric cardia or body of the stomach) and 22 had no a family history of any cancer (Table IGo).


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Table I. Demographics, clinical/pathological characteristics, cancer lifestyle risk factorsa and allelic loss of ESCC patients
 
After obtaining informed consent, patients were interviewed to obtain information on demographic and cancer lifestyle risk factors, including tobacco use, frequencies of alcohol, pickled vegetable and scalding hot food consumption and a detailed family history of cancer (including all cancers in the first, second and third degree relatives). Data were also recorded concerning the clinical/pathological characteristics of the patients' tumors, including location (upper, middle or lower third), pathological grade (G1, well differentiated; G2, moderately differentiated; G3, poorly differentiated), pathological TNM stage (I–IV) and lymph node metastasis (yes or no).

Biological specimen collection and processing
Ten milliliters of venous blood were taken from each patient prior to surgery and genomic DNA was extracted and purified. Tumor tissue obtained during surgery was fixed in ethanol and embedded in paraffin.

Laser microdissection and extraction of DNA
Tumor cells were obtained by laser capture microdissection (LCM) (Pixcell 100; Arcturus Engineering, Mountain View, CA) using methods previously described (23,24). Briefly, unstained, ethanol-fixed, paraffin-embedded 5 µm histological tissue sections were prepared on glass slides, deparaffinized twice with xylene, rinsed twice with 95% ethanol, stained with eosin and air dried. Specific cells of interest were selected from the eosin-stained slides and microdissected by LCM. The cells obtained were immediately resuspended in an 80 µl solution containing 0.01 M Tris–HCl, 1 mM EDTA, 1% Tween-20 and 0.1 mg/ml proteinase K (pH 8.0) and incubated for two nights at 37°C. The mixture was then boiled for 5 min to inactive the proteinase K. Two microliters of this solution were used for each PCR reaction.

Markers, PCR and LOH reading and interpretation
Thirty polymorphic microsatellite markers on chromosome 17p, with heterozygosity ranging from 50 to 93%, were used for this study (Human MapPairs; Research Genetics, Huntsville, AL) (Table IIGo). Of the 30 markers, 13 are found on the physical map (http://www.ncbi.nlm.nih.gov/ ) and 17 are on the genetic map located at 17p13–p11.


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Table II. Summary of frequency of allelic loss in ESCC patients with/without a family history of UGI cancer
 
DNA extracted from tumor cells microdissected from the resection specimen and genomic DNA extracted from venous blood were used for each patient. PCR reactions were carried out using a 10 µl final volume containing 1.0 µl of 10x PCR buffer I (100 mM Tris–HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2), 1.0 µl of 1.25 mM dNTP, 2 µl of DNA extraction buffer, 0.6 µl of each primer, 0.09 µl of AmpliTaq DNA polymerase (Perkin Elmer) and 1 µCi [{alpha}-32P]dCTP. Typical PCR conditions were as follows: 10 min denaturation at 94°C, then 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. An elongation step at 72°C for 10 min was added to the final cycle. The PCR products were mixed with 5 µl of formamide loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol), denatured for 6 min at 95°C and chilled on ice until being loaded onto a 6% polyacrylamide gel. Samples were electrophoresed at 60 W for 1–3 h and radiographed for 1–2 days using Kodak BioMax MR film.

LOH was defined as either complete or nearly complete loss of a band in the tumor sample relative to the corresponding normal DNA (Figure 1Go). There was no convincing evidence of a homozygous deletion in any tumor sample at any of the 30 markers employed. The results were reviewed independently by three investigators (J.Huang, N.Hu and A.M.Goldstein). Discrepant cases were re-evaluated, repeated if necessary and the data were accepted and included in the analysis only if all three reviewers agreed on the results.





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Fig. 1. Allelic loss on markers D17S849 (a), D17S1832 (b) and D17S1176 (c). N, normal DNA from blood; t, DNA from tumor cells.

 
Calculation of the frequency of allelic loss
The frequency of allelic loss at each chromosome locus was calculated as the number of tumors with allelic loss at that locus divided by the number of informative tumors at that locus. The frequency of allelic loss at each chromosome locus was classified as low (0–24%), medium (25–49%), high (50–74%) or very high (>=75%).

Statistical analysis
All statistical analyses were performed using Statistical Analysis Systems (SAS Corp., NC). The t-test (for continuous variables) and {chi}2 or Mantel–Haenszel {chi}2 or Fisher's exact test (for nominal variables) were used for statistical analysis of the relationship between LOH and lifestyle risk factors as well as clinical/pathological characteristics. All P values were two-sided and considered statistically significant at P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient characteristics
A total of 56 ESCC patients, including 34 males and 22 females, were evaluated (Table IGo). Patients with a family history of UGI cancer included 24 patients with cancer in a first degree relative, eight with cancer in a second degree relative and two with cancer in a third degree relative. Patients without a family history of UGI cancer included 22 patients without a family history of any cancer. There were no significant differences between patients with and without a family history of UGI cancer for age (53 versus 54 years, P = 0.31), gender (59 versus 64% male; P = 0.78), tumor location (lower, 21 versus 14%; middle, 71 versus 86%; upper, 8 versus 0%; P = 0.26), tumor grade (G1, 12 versus 9%; G2, 82 versus 77%; G3, 6 versus 14%; P = 0.60), tumor stage (100 versus 91%, stage III; P = 0.15), lymph node metastasis (yes, 28 versus 55%; P = 0.09), tobacco use (44 versus 64%; P = 0.15), alcohol consumption (53 versus 64%; P = 0.43), any pickled vegetable consumption (82 versus 95%; P = 0.15) or very hot food consumption (71 versus 82%; P = 0.34).

Allelic loss in ESCC patients
Allelic loss was detected in 55 of 56 patients (98%) at one or more loci on 17p. Eighteen percent of patients (n = 10) had LOH at 100% of informative markers, half the patients showed loss for >=90% of markers (including 14 with retention of just a single informative marker) and four-fifths of the patients had allelic loss at >50% of the informative markers (Table IGo).

Deletion regions
Of the 30 markers covering the entire short arm of chromosome 17, 19 had a very high frequency of LOH (>=75%), nine had a high frequency of LOH (50–74%) and two markers located at 17p11.1 showed medium frequency of allelic loss (25–49%) (Table IIGo). Indeed, all 28 markers on 17p13.3–p11.2 showed allelic loss in >=65% of informative tumors, implying a very high degree of genetic instability throughout this entire area.

Within this area of high genome instability, however, smaller regions with especially high frequency LOH could be identified. Using the 13 markers located on the physical map, there were three regions where adjacent markers were deleted in >=80% of the informative tumors (Figure 2Go). Deletion region I displayed allelic loss frequencies of 83, 86 and 81% at markers D17S849, D17S926 and D17S1828, respectively, a distance of at least 9 cM or 3.2 Mb on 17p13.3–p13.2. (As D17S849 was our most telomeric marker, the telomeric boundary was not identified.) Deletion region II spanned only 1.67 cM or 0.313 Mb on 17p13.2–p13.1 and included markers D17S938 and TP53, which showed frequencies of 84 and 80%, respectively. Deletion region III included markers D17S804, D17S945 and D17S799, with LOH frequencies of 88, 84 and 90% and covered 11 cM or 1.868 Mb on 17p13.1–p12.



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Fig. 2. Deletion map for chromosome 17p13.3–p11.1 in esophageal squamous cell carcinoma patients.

 
Cancer lifestyle risk factors, clinical/pathological characteristics and LOH
A higher frequency of LOH was observed in patients with a family history of UGI cancer at 19 markers, but for only one of these markers (D17S1854) was this difference statistically significant (92 versus 44%; P = 0.046) (Table IIGo). A higher LOH frequency at D17S926 (in deletion region I) was seen in male (73%) compared with female patients (27%) (P = 0.046). Pickled vegetable consumption was associated with a significantly higher LOH frequency at D17S849 (in deletion region I) (89 versus 0%; P = 0.023). Also, scalding hot food consumption was associated with a significantly lower frequency of allelic loss at D17S1288 (18 versus 75%; P = 0.007). There were no significant associations between tobacco use or alcohol consumption and higher LOH frequencies at any of the 30 markers tested. In addition, none of the four clinical/pathological characteristics examined was associated with a higher frequency of allelic loss at any of the 30 markers.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study reports the pattern of allelic loss found at 30 microsatellite markers on chromosome 17p13–p11 in 56 esophageal squamous cell carcinomas from a high risk population in north central China. Our results indicate that LOH is widespread in this chromosomal region in most patients. Median LOH in our patients was 89%, compared with 43–65% reported in other studies (16). Potential explanations for our higher LOH rates include: the use of LCM tumor samples, which contain purer tumor DNA in which LOH is more easily seen; the possibility that tumors in our study were more advanced than in other studies; the possibility that there truly is greater genetic instability in ESCCs from this high incidence population. Although the number of studies and markers evaluated for LOH on 17p to date is small, the most likely explanation for our higher LOH rate would seem to be our use of LCM samples. Stage differences seem less likely since all reported ESCC LOH studies have analyzed specimens from patients who had been operated on and in the very few studies that examined ESCC from US patients without LCM, LOH has been comparable with studies from Asia (in the range of 60%).

Several tumor suppressor genes and candidate tumor suppressor genes are located on 17p and genetic alterations of these genes have been detected in many human tumors (16,22,25,2729). Our results showed that all markers on 17p13.3–p11.2 had a high frequency of LOH, indicating that almost the entire short arm of chromosome 17 was very unstable in these ESCC patients. Within this broad area of genetic instability, however, we identified three smaller regions at 17p13.3–p13.2, 17p13.2–p13.1 and 17p13.1–p12 with especially high frequencies of LOH.

Several tumor suppressor genes have been described in our deletion region I (primarily 17p13.3). Phillips et al. reported that 80% of 57 early ovarian cancers showed LOH at 17p13.3 (25) and Schultz et al. identified two candidate tumor suppressor genes, OVCA1 and OVCA2, at 17p13.3 (26). HIC-1 (hypermethylated in cancer 1), another putative tumor suppressor gene at 17p13.3, has been shown to be hypermethylated in a number of cancers (27,28). Fujii et al. found LOH in the telomeric portion of 17p in 67% of the breast cancers they examined and determined that this correlated strongly with methylation of HIC-1, suggesting that loss of an unmethylated HIC-1 allele may contribute to the inactivation of HIC-1 protein in cells with a pre-existing methylated allele (29). Recently, Dunn et al. reported allelic loss at D17S849 (on 17p13.3) in Barrett's esophageal adenocarcinoma (22). The very high frequencies of LOH that we found in both markers (D17S849 and D17S926) tested at 17p13.3 raises the possibility that OVCA1, OVCA2 and/or HIC-1 may be important in the development of ESCC in our patients. In addition, we also found a high frequency of LOH in the next adjacent marker (D17S1828), suggesting that an unknown tumor suppressor gene downstream from these previously described genes may also be important.

The principal known tumor suppressor gene in our deleted region II (17p13.2–p13.1) is p53. Dunn et al. also found a high frequency of LOH at the p53 locus in esophageal adenocarcinomas (22) and suggested three possible interpretations for the observed loss: that p53 is the sole target, a shared target or not a target at all. Further analyses of our samples will be required before we can more fully address these potential interpretations.

Our deletion region III at 17p13.1–p12, centromeric to p53, contains several interesting genes: ZNF18 (zinc finger protein 18), ZNF29 (zinc finger protein 29), ALDH3 (aldehyde dehydrogenase 3) and ALDH10 (aldehyde dehydrogenase 10). Allelic loss in this region may affect or reflect the activation or inactivation of these genes or it may be somehow related to p53. To the best of our knowledge, high frequency allelic loss in this region has not previously been reported in esophageal cancer.

There was no strong association between patterns of allelic loss on chromosome 17p and cancer lifestyle risk factors of our patients or the clinical/pathological characteristics of their tumors. Only one of 30 markers was significantly associated with a family history of UGI cancer, male gender, pickled vegetables consumption and eating scalding hot food, and no markers were significantly associated with smoking, alcohol consumption, tumor location, pathological grade, clinical stage or lymph node metastasis. Given the large number of evaluations, the significant associations may have occurred by chance alone.

In summary, we found high frequencies of LOH in nearly all of the 30 microsatellite markers we evaluated on chromosome 17p13.3–p11.2 in 56 ESCCs from a high risk population in northern China. The highest frequencies of allelic loss were found in three smaller regions, which may help us narrow the search for major tumor suppressor genes involved in the development of ESCC.


    Notes
 
4 To whom correspondence should be addressed Email: phil_taylor{at}nih.gov Back

* The first two authors contributed equally to this work. Back


    Acknowledgments
 
The authors thank Duminda Ratnasinghe and Karen Woodson for their helpful suggestions.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 11, 2000; revised June 30, 2000; accepted July 5, 2000.