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Keywords:

  • epithelial ovarian cancer;
  • haplotype;
  • p16;
  • single nucleotide polymorphism;
  • susceptibility

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

p16 is an important tumor suppressor gene, which is inactivated in many kinds of tumors. The common variants of p16 may be associated with the risk of certain tumors development. We analyzed the frequency of two adjacent polymorphisms in p16 exon 3 (540C[RIGHTWARDS ARROW]G and 580C[RIGHTWARDS ARROW]T) and their haplotype in blood samples from epithelial ovarian cancer (EOC) patients and healthy controls using polymerase chain reaction–restriction fragment length polymorphism. The results showed that the genotype frequency of p16 580C[RIGHTWARDS ARROW]T polymorphism was significantly different among histologic subtypes of EOC (P= 0.02). T allele carriers significantly reduced the risk of serous EOC; the adjusted odds ratio was 0.40 (95% CI = 0.19–0.84). There are neither association between p16 540C[RIGHTWARDS ARROW]G polymorphism and EOC development, progression, nor association between the haplotypes of two single nucleotide polymorphisms and the tumor development. Our results suggested that the p16 580C[RIGHTWARDS ARROW]T polymorphism might affect the individual susceptibility to specific subtypes of EOC. Different types of ovarian cancer might adopt distinct carcinogenetic pathways. However, this result may be further validated in a larger sample of patients.

Ovarian cancer is the third most common gynecological cancer, which is the leading cause of cancer-related death among females in China today(1). Because of lack of symptoms from this silent disease, ovarian cancer might have spread to the abdomen in about 70% of cases by the time it was detected and, unfortunately, most patients die within 5 years. Many histologic types of ovarian tumors have been described. However, more than 70% of ovarian malignant tumors are epithelial tumors. Although the etiology of ovarian cancer is still unclear, it is clear that multiple genetic changes have been verified, including activation of protooncogenes and inactivation of tumor suppressor genes.

p16 (also known as CDKN2/MTS-1/INK4a) is an important tumor suppressor gene located on chromosome 9p21 and is composed of three exons encoding a 156 amino acid protein. The p16 gene product can bind to CDK4 and CDK6 and inhibits their interaction with cyclin D1. The inhibition of cyclin D1–CDK4/6 complex activity prevents retinoblastoma protein phosphorylation and the release of E2F and then leads to the inhibition of cell cycle in G1/S transition(2). The inactivation of p16 results in increasing cell proliferation and is thought to contribute to malignancy. The aberrant p16 protein caused by point mutations, small deletions, large hetero- and homozygous deletions, and silencing by methylation of CpG islands in the promoter region has been found in many kinds of human tumors, indicating that these factors are closely related to tumor genesis(3–7). In ovarian cancer, p16 deletion occurred at a rate of 50% in cell lines, and the loss of p16 expression was detected in 11–37% of studied cases(8–11). Havrilesky et al.(12) also showed that the loss of expression of the p16 tumor suppressor occurred more often in ovarian cancers lacking p53 mutations. It was supported by some studies that the hypermethylation of p16 promoter may be a mechanism underlying the downregulation of p16 gene and is associated with the development and progression of ovarian cancer(13–15).

However, the polymorphisms in the coding region and 3′ untranslated region (UTR) of p16 have been associated with the development and progression of certain cancers in previous studies(16,17). Although it has been shown that some polymorphisms were related to the altered protein function of p16, others were not. It has been unclear to date whether the polymorphisms in p16 gene are related to the risk of ovarian cancer development and progression. In the present paper, we discussed the relationship between two adjacent polymorphisms (540C[RIGHTWARDS ARROW]G and 580C[RIGHTWARDS ARROW]T), which were identified recently in the 3′ UTR of exon 3 of p16, and the risk of epithelial ovarian cancer (EOC) development and progression.

We hypothesize that two genetic polymorphisms in the p16 gene are responsible for interindividual variation in the production of p16 and in turn leads to individual susceptibility to EOC. To test this hypothesis, we conducted a hospital-based case–control study on patients with EOC and control women to evaluate the association between the p16 genotypes/haplotypes and the risk of EOC development and progression in North China.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Subjects

Two hundred and five patients with ovarian cancer admitted for tumor resection were selected in the Fourth Affiliated Hospital, Hebei Medical University from January 2001 to December 2005. The EOC was identified through histopathologic examination by the Department of Pathology of the same hospital. In all the ovarian cancers, histology types included 81 serous, 21 mucinous, 69 endometrioid, and 34 undifferentiated. Clinical stage of the cases was according to FIGO, 89 (43.4%) were I–II stage and 116 (56.6%) were III–IV stage. The control group consisted of 268 women in reproductive age without any malignant disease confirmed by surgical exploration at voluntary abortion, cesarean section, or pathologically confirmed after hysterectomy for dysfunction uterine bleeding. All women in control group had no previous oophorectomy and history of cancer or genetic disease. The mean age of the patients and controls was 51 years (range 20–75 years) and 48 years (range 20–73 years), respectively. All the cancer patients and control subjects were unrelated and of Han nationality from Shijiazhuang city and surrounding regions. The study was approved by the Ethics Committee of Hebei Cancer Institute, and informed consent was obtained from all recruited subjects.

DNA extraction

Venous blood (5 mL) from each subject was collected into Vacutainer tubes containing ethylenediamine tetraacetic acid and stored at 4°C. After sampling, genomic DNA was extracted within 1 week through digesting with proteinase K (Merck, Darmstadt, Germany) followed by a salting out procedure according to the method published by Miller et al.(18).

Determination of genotypes

The p16 540C[RIGHTWARDS ARROW]G and 580C[RIGHTWARDS ARROW]T genotypes were determined by polymerase chain reaction (PCR)–restriction fragment length polymorphism assay. The primers for amplifying the p16 540C[RIGHTWARDS ARROW]G and 580C[RIGHTWARDS ARROW]T fragment were 5′-GATGTGCCACACATCTTTGACCT-3′ (forward) and 5′-CTACGAAAGCGG GGTGGGTTGT-3′ (backward). The PCR was performed in a 25-μL volume containing 100 ng of DNA template, 2.5 μL of 10 × PCR buffer, 2.0 mM of MgCl2, 2.5 U of Taq DNA polymerase (BioDev-Tech., Beijing, China), 250 μM of deoxyribonucleoside triphosphate (dNTPs), and 250 nM of each primer. The PCR cycling conditions were 10 min at 94°C followed by 35 cycles of 45 sec at 94°C, 45 sec at 62°C, and 1 min at 72°C, and with a final step at 72°C for 7 min to allow for the complete extension of all PCR fragments. As a result, PCR product was 181 bp. Two 8 μL aliquot of PCR product were subjected to digestion at 37°C overnight in a 10 μL reaction containing 10 U of MspI (SBS Genetech Co., Ltd, Beijing, China) or 10 U of HaeIII (TaKaRa Biotechnology Co., Ltd, Dalian, China), respectively. After digestion, the products were separated on a 3% agarose gel stained with ethidium bromide. For p16 540C[RIGHTWARDS ARROW]G, the 540C alleles were represented by DNA bands with size of 104 and 77 bp, the 540G alleles were represented by a DNA band with size of 181 bp, whereas the heterozygotes displayed a combination of both alleles (181, 104, and 77 bp) (Fig. 1). For p16 580C[RIGHTWARDS ARROW]T, the 580C alleles were represented by DNA bands with size of 142 and 39 bp, the 580T alleles were represented by a DNA band with size of 181 bp, whereas the heterozygotes displayed a combination of both alleles (181, 142, and 39 bp) (Fig. 2).

image

Figure 1. p16 C540G genotyping by PCR–RFLP analysis followed by separation on 3% agarose gel as described in text. Lane M = 100 bp ladder; lane 1, 2, 6, 7 = C/C; lane 3, 4 = C/G; lane 5 = G/G; lane 8 = PCR product.

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image

Figure 2. p16 C580T genotyping by PCR–RFLP analysis followed by separation on 3% agarose gel as described in text. Lane M = 100 bp ladder; lanes 3, 5 = C/C; lane 1, 2 = C/T; lane 4 = T/T; lane 6 = PCR product.

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Statistical analysis

Statistical analysis was performed using SPSS 10.0 software package (SPSS Company, Chicago, IL). Hardy–Weinberg analysis was performed to compare the observed and expected genotype frequencies using the χ2 test. Comparison of the p16 540C[RIGHTWARDS ARROW]G and 580C[RIGHTWARDS ARROW]T genotype distribution in the test groups was performed by means of two-sided contingency tables using χ2 test. The p16 540C[RIGHTWARDS ARROW]G and 580C[RIGHTWARDS ARROW]T haplotype frequencies and linkage disequilibrium coefficient were estimated using the EH linkage software (version 1.2, Rockefeller University, New York, NY). The odds ratio (OR) and 95% confidence interval were calculated using an unconditional logistic regression model and adjusted by age accordingly. A probability level of 5% was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

p16 gene polymorphisms with susceptibility to EOC

Table 1 shows the distribution of p16 genotypes in the ovarian cancer patients and the controls.

Table 1.  Distribution of genotypes and alleles of two single nucleotide polymorphisms in p16 genes
GeneGenotype/allelePatients number (frequency)Controls number (frequency)P2 test)
p16C540G
 GenotypeG/G1 (0.5)2 (0.7)0.45
G/C11 (5.4)22 (8.2) 
C/C193 (94.1)244 (91.0) 
 AllelotypeG13 (3.2)26 (4.9)0.19
C397 (96.8)510 (95.1) 
p16C580T
 GenotypeT/T0 (0)3 (1.1)0.29
T/C44 (21.5)61 (22.8) 
C/C161 (78.5)204 (76.1) 
 AllelotypeT44 (10.7)67 (12.5)0.40
C366 (89.3)469 (87.5) 

The distribution of the p16 540C[RIGHTWARDS ARROW]G and 580C[RIGHTWARDS ARROW]T polymorphisms’ genotypes in the controls did not deviate significantly from those expected under Hardy–Weinberg equilibrium.

The C and G allele frequencies of p16 540C[RIGHTWARDS ARROW]G in the ovarian cancer patients had no significant difference from that in the controls (P= 0.45). The distribution of genotypes (C/C, C/G, G/G) in the ovarian cancer patients was similar to that in the controls (P= 0.19). By contrast, G allele carriers (G/G + G/C genotype) did not significantly modify the risk of ovarian cancer compared with C/C genotype; the adjusted OR was 0.63 (95% CI = 0.31–1.30) (Table 2).

Table 2.  Association of two single nucleotide polymorphisms in p16 gene with the risk of EOC development
GeneGenotypeControlsPatientsOR (95% CI)
  • a

    The OR of the C/G + G/G genotype against the C/C genotype in ovarian cancer.

  • b

    The OR of the C/T + T/T genotype against the C/C genotype in ovarian cancer.

p16C540GC/C244 (91.0)193 (94.1) 
C/G + G/G24 (9.0)12 (5.9)0.63 (0.31–1.30)a
p16C580TC/C204 (78.5)161 (76.1) 
C/T + T/T64 (21.5)44 (23.9)0.84 (0.53–1.36)b

The C and T allele frequencies of p16 580C[RIGHTWARDS ARROW]T did not differ between the ovarian cancer patients and the controls (P= 0.29). The distribution of genotypes (C/C, C/T, T/T) was not significantly different between cases and controls (P= 0.40). Compared with C/C genotype, T/T in combination with the C/T genotype did not significantly modify the risk of ovarian cancer; the adjusted OR was 0.84 (95% CI = 0.53–1.36) (Table 2).

Haplotype of p16 two single nucleotide polymorphisms with susceptibility to EOC

The results of the haplotype analyses are shown in Table 3. The frequency of 540G/580T haplotype was 0% in women of North China. The most common haplotype in the controls was 540C/580C (82.7%), followed by the 540C/580T (12.4%), and 540G/580C (4.8%). Compared with the most common haplotype (540C/580C), neither the 540C/580T nor the 540G/580C haplotype significantly modified the risk of ovarian cancer; the adjusted OR was 0.82 (95% CI = 0.55–1.27) and 0.63 (95% CI = 0.32–1.24). The C allele of p16 540C[RIGHTWARDS ARROW]G and C allele of p16 580C[RIGHTWARDS ARROW]T showed high linkage disequilibrium of inline image

Table 3.  Haplotypes of p16C540G and p16C580T with the risk of EOC development
HaplotypesControlsOvarianOR (95% CI)a
  • a

    OR of the 540C/580T haplotype against the 540C/580C haplotype in ovarian cancer.

  • b

    OR of the 540G/580C haplotype against the 540C/580C haplotype in ovarian cancer.

540C/580C443 (82.7)353 (86.1) 
540C/580T67 (12.4)44 (10.7)0.82 (0.55–1.27)a
540G/580C26 (4.8)13 (3.2)0.63 (0.32–1.24)b
540G/580T0 (0)0 (0) 

p16 single nucleotide polymorphism with susceptibility to different histologic subtypes of EOC

The genotype frequency of p16 540C[RIGHTWARDS ARROW]G was not significantly different among histologic subtypes of EOC (P= 0.68), but the distribution of p16 580C[RIGHTWARDS ARROW]T polymorphism was significantly different among histologic subtypes (P= 0.02). The T/C genotype frequency of serous was lower than endometrioid and undifferentiated. The frequency of T allele among serous, mucinous, endometrioid, undifferentiated, and controls was 5.5%, 9.5%, 15.2%, 14.7%, and 12.5%, respectively. There is a significant difference between T allele frequency of serous and those of endometrioid, undifferentiated, and controls (all P < 0.05). Compared with the controls, T allele carriers significantly reduced the risk of serous EOC; the adjusted OR was 0.40 (95% CI = 0.19–0.84). T allele carriers were not associated with risk of mucinous, endometrioid, and undifferentiated; the adjusted OR was 0.75 (95% CI = 0.24–2.31), 1.55 (95% CI = 0.78–2.50), and 1.38 (95% CI = 0.60–2.92), respectively.

p16 gene polymorphisms with susceptibility to different clinical stage of EOC

The genotype frequencies of p16 540C[RIGHTWARDS ARROW]G and 580C[RIGHTWARDS ARROW]T polymorphisms were not significantly different between clinical stages I–II and stages III–IV (Table 4). The 540G and 580T may not be associated with progression of ovarian cancer. Compared with the controls, 540G allele carriers could not significantly enhance progression of EOC; the adjusted OR was 0.61 (95% CI = 0.22–1.64) for clinical stage I–II patients and it was 0.65 (95% CI = 0.27–1.56) for clinical stages III–IV. The 580T allele carriers were not associated with progression of EOC; the adjusted OR was 0.70 (95% CI = 0.38–1.29) for clinical stage I–II patients and 1.01 (95% CI = 0.61–1.69) for clinical stages III–IV.

Table 4.  Distribution of genotypes and alleles of two single nucleotide polymorphisms in p16 genes among different histologic subtypes of EOC
GeneGenotypeSerousMucinousEndometrioidUndifferentiatedI–II stageIII–IV stage
  • a

    χ2 test among different histologic subtypes of EOC.

  • bχ2 test among different clinical stage of EOC.

p16C540G
 G/G + G/C513357
C/C7620663184109
P2 test) 0.87a 0.68b 
p16C580T
 T/C9421101628
C/C721748247388
P2 test) 0.02a 0.28b 

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

In this paper, we study the role of polymorphisms at 3′ UTR of p16 in the risk of EOC development and progression. Our results showed that the 540C[RIGHTWARDS ARROW]G polymorphism of p16 was not associated with the risk of EOC development and progression. But the 580C[RIGHTWARDS ARROW]T polymorphisms were associated with the development of different histologic subtypes of EOC. The rare allele (T allele) was associated with a lower risk of serous EOC: women having T allele had a 60% reduction in the risk. These results might partly support our hypothesis. Further, there were no associations between the haplotypes of 540C[RIGHTWARDS ARROW]G with 580C[RIGHTWARDS ARROW]T polymorphism and the risk of EOC development, although the C allele of p16 540C[RIGHTWARDS ARROW]G and C allele of p16 580C[RIGHTWARDS ARROW]T showed high linkage disequilibrium in the women of North China.

The 540C[RIGHTWARDS ARROW]G and 580C[RIGHTWARDS ARROW]T polymorphisms located at the 3′ UTR of exon 3 of p16. Although the function of two polymorphisms in normal tissues was unknown, the study showed that 540C[RIGHTWARDS ARROW]G polymorphism in CDKN2A (p16) gene with 74 C[RIGHTWARDS ARROW]A polymorphism of intron 1 of CDKN2B gene in linkage disequilibrium, the two cosegregating polymorphisms were associated with the lower expression of TP53 protein(19). Moreover, the recent evidence strongly suggested that the 3′ UTR of messenger RNA was involved in regulation of p16 gene expression by controlling nuclear export, polyadenylation status, subcellular targeting, translation rates, and messenger RNA degradation(20). Therefore, those two polymorphisms may have a signification at function. Previous study has indicated that both 540C[RIGHTWARDS ARROW]G and 580C[RIGHTWARDS ARROW]T polymorphisms were associated significantly with the shorter progression time from primary melanoma to metastatic disease(19). The 540C[RIGHTWARDS ARROW]G polymorphism of p16 may be increased linearly with increasing risk in familial melanoma(21), whereas Geddert et al.(22) showed that frequencies of p16 540C[RIGHTWARDS ARROW]G polymorphism in gastric and esophageal adenocarcinomas did not differ significantly from the healthy control group. Therefore, these polymorphisms were unlikely to be associated with the risk of adenocarcinomas of the upper gastrointestinal tract. Zheng et al.(23) also demonstrated that the genotypes of p16 540C[RIGHTWARDS ARROW]G and 580C[RIGHTWARDS ARROW]T polymorphisms or haplotypes might not play a major role in the etiology of squamous cell carcinoma of the head and neck. Unlike the above reports, our results showed that frequencies of p16 580C[RIGHTWARDS ARROW]T polymorphism genotype in serous EOC were significantly different from the other subtypes of EOC. The T allele carriers may have a lower risk of serous EOC development. There is a difference in reports about expression of p16 protein in serous EOC. Langosch et al.(11) showed that p16 aberrant was detected in 71–79% of endometrioid and mucinous, but only in 10% of serous papillary carcinomas. Havrilesky et al.(12) reported that p16 loss was more common in serous (40%) than in nonserous ovarian cancers (17%, P= 0.07). Ichikawa et al.(24) also showed that inactivation of p16/CDKN2 might be the histologic type-specific events involved in ovarian tumorigenesis. Because the role of p16 580C[RIGHTWARDS ARROW]T polymorphism in p16 protein expression is still unclear, future work should focus on studies in a larger population and on its mechanism of regulation.

EOC contains several different histologic subtypes, of which serous type is most common. The others include mucinous, endometrioid, clear cell, Brenner, and undifferentiated carcinoma, etc(25). Our study samples included serous (81% versus 39.5%), endometrioid (69% versus 33.7%), undifferentiated (34% versus 16.6%), and mucinous (21% versus 10.2%) tumors. There are distinct morphologic appearance and subtle clinical differences among different EOC subtypes. The studies showed that the different subtypes might be different in pathogenesis and progression because of a molecular evidence for heterogeneity among them(26). The polymorphism in some genes may be associated with the risk of different histologic subtypes of EOC. For example, GSTM1 null genotypes might increase the risk of endometrioid and clear-cell carcinoma in ovarian cancer(27). The single nucleotide polymorphisms at coding region of follicle-stimulating hormone receptor gene were associated with the great risk of developing serous and mucinous types of ovarian cancers(28). Our result showed that single nucleotide polymorphism at the 3′ UTR of exon 3 of p16 might play a different role in the risk of EOC histologic subtypes, too.

To the best of our knowledge, this is the first study to look for an association between the two polymorphisms in p16(CDKN2) exon 3 and the risk of EOC. In conclusion, our results showed that the carrier of 580T allele may significantly reduce the susceptibility to serous type of EOC. It suggests that there is a possible distinctness in pathogenesis of different histologic subtypes. This result may be further validated in a larger sample of patients.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

We thank Dr Yu Feng-Ling of the Department of Pathology, Hebei Cancer Institute for histologic subtypes of EOC.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
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