1 Department of Biochemistry, Al Quds University; 2 Abu-Dies, Muqased Hospital, East Jerusalem; 6 Augusta Victoria Hospital, Palestinian Authority; 3 Department of Pathology, 4 GI Oncology Unit, 7 Department of Gastroenterology, Tel Aviv Sourasky Medical Center; 8 Tel Aviv University, Tel Aviv; 9 Department of Mathematics, Technion-Israel Institute of Technology, Haifa, Israel; 10 Middle East Cancer Consortium, Israel; 5 Department of Pathology, Columbia University, New York, NY, USA
Received 29 October 2001; revised 14 December 2001; accepted 9 January 2002
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Abstract |
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To evaluate and compare differences in the molecular genetics among high-risk (Ashkenazi Jews), intermediate-risk (Sephardic Jews) and low-risk (Palestinians) groups for colorectal cancer who live in the same geographical region.
Patients and methods:
The 19951996 records from the Tel Aviv Medical Center and Muqased hospital (East Jerusalem) randomly identified patients with colorectal cancer. There were 25 patients from each ethnic group. Epidemiological data were obtained from interviews with the patients and from their hospital charts. The levels of cyclin D1, ß-catenine, p27, p53, Ki-67 and Her-2/neu proteins were determined by immunohistochemistry. The main outcome measures were the association between gene expression and colorectal incidence in the different ethnic groups.
Results:
Ashkenazi Jews have the highest rate of colorectal cancer, and are diagnosed at an early stage compared with Palestinians (72% and 33% of the cases are in Dukes A and B, respectively), and, hence, this may explain the better 5-year survival rate among this group. Sephardic Jews are diagnosed at a more advanced stage, the tumors are poorly differentiated and they lack p27. Palestinians have significantly higher cyclin D1 levels. There was a statistically significant inverse correlation between the expression of ß-catenine and cyclin D1, as well as p53 and p27 (P <0.05).
Conclusions:
Increased expression of cyclin D1, p53, Ki-67, ß-catenine and Her-2/neu, and decreased expression of p27 may be important events in the three ethnic groups with colorectal cancer. The lower mortality rate among Ashkenazi Jews may be partially explained by their better molecular biology profile.
Key words: Ashkenazi Jews, colorectal cancer, molecular epidemiology, Palestinians, Sephardic Jews
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Introduction |
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Among Jews in Israel, CRC is the leading cause of death from cancer [7] while the Palestinian population appears to be protected from it [7]. It has long been noted that Israelis of European origin are more susceptible to CRC then AsianAfrican born Jews (42 per 100 000 versus 25 per 100 000, mean age standardized rates for 19891993) [7]. Studies from the United States and Australia have also corroborated the findings that Jewish populations, particularly those of European origin, are at high risk for CRC [8]. The incidence of the disease among Palestinians is estimated to be 7 per 100 000, approximately one-fifth that of Jews in Israel [7].
The genetic makeup of Jews and Arabs is almost identical: the two groups share common prehistoric ancestors and have a common geographical and ecological milieu. Israel is therefore an ideal laboratory to test these concepts because of a well defined population of subgroups that have retained their ethnicity. The intra-population variations in incidence, morbidity and mortality suggest that at least some genetic differences partially account for these obvious differences in the degree of susceptibility to CRC. Furthermore, there is a genetic susceptibility for germline predominant mutations among the Jewish population for developing breast (BRCA 1, 2 mutations) and colon (APC) cancers, which are known in the literature as the Ashkenazi Jewish mutations. It is also possible that lifestyle differences, and in particular, differences in diet, may be a protecting factor among Palestinians (as in the economically underdeveloped world), and a risk factor among the Ashkenazi Jews (as in the economically developed world).
These patterns of biological markers associated with CRC have not yet been evaluated prospectively in a well-designed study based on different ethnic populations. This is the first study to do so, setting out to define the molecular differences in CRC patients from highly susceptible Ashkenazi Jews, intermediate-risk Sephardic Jews and low-risk Palestinians.
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Material and methods |
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Immunohistochemistry was used to determine levels of cyclin D1, ß-catenine, p27, p53, Ki-67 and Her-2/neu proteins.
Immunohistochemistry
All immunohistochemical analyses utilized the avidinbiotin complex immunoperoxidase technique (Figure C1). Tissue sections (5 µm) were mounted on poly-L-lysine-coated slides. After deparaffinization in Americlear (Baxter, McGaw Park, IL, USA) and absolute ethanol, sections were hydrated through a series of graded alcohol, distilled water, and phosphate-buffered saline at pH 7.4. The slides were immersed in 10 mM citrate buffer (pH 6) and heated by microwave to enhance antigen exposure for a total of 10 min. Potential background signals were blocked using goat or horse serum for 20 min, and primary anti-human antibodies were added and incubated in a high-humidity chamber. These included mouse monoclonal antibodies to cyclin D1 (2 h at 25°C), p53 (overnight at 4°C), and Ki-67 (1 h at 25°C) (Immunotech, Inc., Westbrook, ME, USA), ß-catenine (2 h at 25°C) and p27Kip1 (1 h at 25°C) (Transduction Laboratories, Lexington, KY, USA), and Her-2/neu (1 h at 25°C) (Oncogene Research Products, Cambridge, MA, USA). Although all the concentrations of primary antibodies gave good nuclear staining, the optimal concentrations that produced a minimal background were: a 1:200 dilution for cyclin D1 and p27; a 1:5 dilution for p53; a 1:75 dilution for ß-catenine; a 1:400 dilution for Ki-67; and a 1:50 dilution for Her-2/neu. Positive controls included breast adenocarcinoma for cyclin D1, Her-2/neu, Ki-67 and p53, gastric adenocarcinoma for p27, and colon adenocarcinoma for ß-catenine.
Subsequently, the Vectastain Elite ABC kit was used (Vector Laboratories, Burlingame, CA, USA) according to the manufacturers instructions. Color development was accomplished with a 0.375 mg/dl solution of a 3,3'-diamino-benzidine tetrahydrochloride (Sigma Chemical Co., St Louis, MO, USA) containing 0.003% hydrogen peroxide. The specificity of the antibodies was demonstrated by inhibition of immunohistochemical staining in positive controls, by pre-incubating the antibody with 1 mg of the immunizing peptides for 1 h at 40°C, representing about a 100-fold excess of peptide over antibody.
Interpretation of immunohistochemical staining
An experienced surgical pathologist (L.E.T.) interpreted the staining. Nuclear staining was considered positive if the chromogen was clearly detected in at least 10% of the nuclei within a microscopic field. Immunoreactivity was considered positive if the chromogen was detected in at least 5% of the nuclei within a microscopic field. Staining intensity included four scales: no staining (scale level 0); weakly positive and comparable with adjacent non-neoplastic epithelium (scale 1); moderately positive (scale 2); and strongly positive (scale 3). Scales 0 and 1 were regarded as negative and scales 2 and 3 as positive. Positive and negative controls were included within each batch of slides. To confirm reproducibility, 25% of the slides were chosen at random and another level of the same tissue was stained once again in the same batch. All batches were coded and blindly scored twice. Duplicate slides gave similar results.
Statistical analysis
The proportions of samples expressing the different genes from different histological categories were computed, and then compared across categories for selected factors (including gender, age, cigarette smoking, alcohol consumption, differentiation, Dukes stage, and mortality). When comparing proportions positive for staining across histological categories, Fishers exact test and chi-squared tests were employed [9]. McNemars test [10] was used for testing asymmetry in the association between the different genetic markers.
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Results |
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Discussion |
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As in other published series [1, 1118], increased expression of cyclin D1, p53, Ki-67, ß-catenine and Her-2/neu, as well as decreased expression of p27 are all important events that may play a role in patients prognosis. Thus, cyclin D1 amplification [17], increased expression of cyclin D1 in tumors from the entire gastrointestinal tract [1820], and decreased expression of p27 in gastric [20] and colonic adenocarcinomas [21] are associated with a poorer prognosis.
Ashkenazi Jews have the highest rate of CRC, yet the 5-year survival rate was significantly better among this group (70%) compared with Sephardic Jews (47%) and Palestinians (30%). The better 5-year survival rate is probably due to the early diagnosis of the disease among Ashkenazi Jews (72% in Dukes A and B) as compared with only 33% among Palestinians. The comparisons of the immunohistochemical data among the three groups do not compare like with like, since the CRC comparison among the different groups was not of the same stage or grade. It is possible that Palestinians and Sephardic Jews show a poorer 5-year survival because 67% and 50% of their tumors, respectively, were at a more advanced stage compared with 28% of tumors in the Ashkenazi group. This difference in survival rate can therefore be attributed to earlier stage at the time of diagnosis. Indeed, screening programs for early detection of CRC are more popular among Ashkenazi Jews than among Sephardic Jews, whereas screening in fact does not exist among Palestinians. At the same time a significant decrease in the level of p27 among Sephardic Jews and a significant increase in cyclin D1 in Palestinians may partly contribute to the poorer prognosis among these groups.
It was recently demonstrated that ß-catenine regulates the expression of cyclin D1 [22]; however, in our series there was an inverse relationship in their expression. There might be other factors, still undetermined, that regulate the interaction between these two genes.
An interesting finding is the very high rate (92%) of well differentiated tumors among Sephardic Jews, as compared with Ashkenazi Jews (32%) and Palestinians (38%) (P <0.05). These findings have not been described previously. This difference might be an artifact of multiple comparisons in a relatively small study population. Since this difference was statistically significant it might be that another, yet unidentified, molecular marker exists in the former and not in the latter groups.
It is not clear from the present data what the bases are for the difference in CRC incidence among the three groups. However, what is indisputable is that noticeable histological and molecular differences were evident in tumors of patients from the three groups. Other epidemiological (and in particular nutrition) and molecular factors are involved, which might further explain the observed differences. The number of patients in each group, however, is too small and the results should be confirmed in larger scale studies.
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Acknowledgements |
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Footnotes |
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References |
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