Abstract
Purpose
Gallbladder cancer is a lethal malignancy of hepato-biliary system with high incidence in North India, especially along gangetic plain. The let-7 microRNAs play a key role in regulating KRAS expression and a polymorphism in 3′ untranslated region (rs61764370, T/G) of KRAS leads to its higher expression. This polymorphism is known to be associated with increased risk and prognosis of various cancers but its association with gallbladder cancer has not been evaluated. To address this research question, we evaluated whether rs61764370 variant is associated with gallbladder cancer susceptibility and clinical outcomes.
Methods
In present case–control study, we enrolled 541 patients with gallbladder malignancy and 307 controls. Genomic DNA was obtained from peripheral blood and genotyping was performed using Taqman allelic discrimination assay.
Results
Heterozygous (TG) individuals are at a significant higher risk for GBC as compared with wild genotype (TT) (p = 0.007, odds ratio = 2.56, 95 % CI 1.27–5.18). At allelic level, allele G has significant higher risk for GBC as compared with T allele (p = 0.008, odds ratio = 2.5, 95 % CI 1.25–5.01). Survival analysis reveals decrease in overall survival for heterozygous genotype (p < 0.0001, hazard ratio = 3.42, 95 % CI 1.21–4.20). Also, significant decrease in overall survival was observed for patient carrying allele G (p < 0.0001, HR = 2.89, 95 % CI 1.21–4.20) as compared with allele C.
Conclusions
We conclude that KRAS rs61764370 polymorphism is significantly associated with risk and prognosis of gallbladder malignancy in this endemic belt.
Keywords: Gallbladder cancer, KRAS, let-7, rs61764370
Introduction
Gallbladder cancer (GBC) is the most common malignancy of the biliary tract with high incidence in Northern India especially along the gangetic belt (Dhir and Mohandas 1999; Manoharan et al. 2010; Kazmi et al. 2014; Yadav et al. 2015; Gupta et al. 2016). Complete surgical resection remains only potential curative treatment for this malignancy but with high recurrence rate. However, at the time of diagnosis most patients have advanced disease due to lack of specific sign and symptoms (Shaffer 2008; Zhu et al. 2010; Dutta 2012). The prognosis of GBC remains extremely poor, because of late presentation and aggressiveness. Hence, novel diagnostic and prognostic biomarkers are needed for better clinical outcome.
MicroRNAs (miRNAs) are short non-coding RNAs of 20–24 nucleotides long that play an important role in gene regulation of their target mRNAs by binding to complementary sites in the 3′ untranslated region (UTR) (Johnson et al. 2005; Hollestelle et al. 2011). A large number of studies have confirmed miRNA binding site polymorphism to be novel class of polymorphic variations of clinical importance and could be useful biomarkers for disease progression, treatment response, and prognosis (Cipollini et al. 2014). Let-7 miRNAs are genetic regulators that are important in controlling KRAS expression and a polymorphism in 3′UTR (rs61764370 T/G) of the gene disrupts let-7 miRNA binding site which ultimately leads to higher expression of ras protein (Chin et al. 2008; Hollestelle et al. 2011). The association of rs61764370 polymorphism with several types of cancer has been extensively studied (Chin et al. 2008; Nelson et al. 2010; Sebio et al. 2013; Hollestelle et al. 2011; Ratner et al. 2010; Christensen et al. 2009). However, its association with risk and prognosis for gallbladder malignancy has not been evaluated. Therefore, in present study we investigated KRAS rs61764370 polymorphism and its association with susceptibility, clinical outcomes, and survival of GBC patients in Northern Indian high risk population.
Material and methods
This present hospital based case control study was ethically approved by the Institutional Ethics Committee and all subjects provide written inform consent before enrolment. In our study, a total of 541 histologically confirmed or fine needle aspiration cytology-proven cases of gallbladder cancer were included. There are 307 controls (healthy subjects, without any malignancy or gallstones or polyp) in this cohort from the same geographical area and ethnicity. Cases and controls were mainly recruited from January 2010 to October 2015 from outpatient department (OPD) of King George’s Medical University, Lucknow. All the procedures were according to the Declaration of Helsinki. Staging for gallbladder malignancy was done according to 7th edition of American Joint Committee on Cancer—Tumor node metastasis classification (TNM), 2010 or by radiological evaluation. Patients were followed after one month of recruitment to their death or completion of the study whichever occurred earlier. Telephonic interview was done to obtain prognostic data (number of chemotherapy cycles and overall survival).
Genomic DNA was obtained from 3 ml peripheral blood (collected in EDTA) which was obtained from each subject by intravenous puncture. Integrity of DNA was checked in 0.8 % agarose gel and quantification of DNA was assessed using nanodrop spectrophotometer. Genotyping was done using Taqman allelic discrimination assay (Applied Biosystems, USA) with a custom-designed primer probe (Forward primer: 5′-GCCAGGCTGGTCTCGAA-3′, reverse primer: 5′-CTGAATAAATGAGTTCTGCAAAACAGGTT-3′, VIC reporter probe: 5′-CTCAAGTGATTCACCCAC-3′, and FAM reporter probe: 5′-CAAGTGATTCACCCAC-3′) set using ABI PRISM 7500 software.
Statistical analysis was performed by Statistical Package for the Social Sciences (SPSS Version 16.0, Chicago, IL) for windows or using Graph pad prism (Version 5.0). Two-tailed p value <0.05 was considered to be statistically significant. Kaplan–Meier survival method was used for patient survival’s estimation and the difference was analyzed by the log-rank test. Hazard of failure was estimated using Cox proportional hazard model.
Results
In the present case–control study, a total of 848 subjects were recruited including 541 cases of gallbladder cancer while 307 subjects were healthy controls. Control group was matched with cases by age and gender. Clinical characteristics of the study population are illustrated in Table 1. For both cases and controls, the observed genotype frequency was in Hardy–Weinberg equilibrium. We observed significant difference in genotype frequency for rs 61764370 polymorphism between GBC cases and healthy population (Table 2). However, we were unable to find any mutant genotype (GG) in our recruited subjects. Individuals having heterozygous genotype (TG) have significant higher risk for GBC as compared with wild genotype (TT) (p = 0.007, odds ratio = 2.56, 95 % CI 1.27–5.18). Similarly, at allelic estimation, allele G has significant higher risk for GBC as compared with T allele (p = 0.008, odds ratio = 2.5, 95 % CI 1.25–5.01).
Table 1.
Table showing clinical characteristics of recruited subjects
| Parameters | Case (n = 541) | Controls (n = 307) | p value |
|---|---|---|---|
| Age ± SD | 50.33 ± 15.7 | 48.99 ± 11.28 | 0.19 |
| Sex | – | ||
| Male/female | 153 (28.3 %)/388 (71.7 %) | 100 (32.57 %)/207 (67.43 %) | 0.22 |
| Stage at diagnosis | N/A | – | |
| 1a/1b | 1 (0.2 %)/7 (1.3 %) | – | |
| 2 | 25 (4.6 %) | – | |
| 3a/3b | 57 (10.5 %)/61 (11.3 %) | – | |
| 4a/4b | 111 (20.5 %)/244 (45.1 %) | – | |
| Missing | 35 (6.5 %) | – | |
| T | N/A | – | |
| 1 | 7 (1.3 %) | – | |
| 2 | 84 (15.5 %) | – | |
| 3 | 165 (30.5 %) | – | |
| 4 | 217 (40.1 %) | – | |
| Missing | 68 (12.6 %) | – | |
| N | N/A | – | |
| 0 | 114 (21.1 %) | – | |
| 1 | 138 (25.5 %) | – | |
| 2 | 137 (25.3 %) | – | |
| Missing | 152 (28.1 %) | – | |
| M | N/A | – | |
| 0 | 285 (52.7 %) | – | |
| 1 | 189 (34.9 %) | – | |
| Missing | 67 (12.4 %) | – | |
| Gallstones | N/A | – | |
| Present/absent | 424 (78.4 %)/96 (17.7 %) | – | |
| Missing | 21 (3.9 %) | – | |
| Jaundice | N/A | – | |
| Present/absent | 337 (62.3 %)/158 (29.2 %) | – | |
| Missing | 46 (8.5 %) | – | |
| Palpable lump | N/A | – | |
| Present/absent | 363 (67.1 %)/151 (27.9 %) | – | |
| Missing | 27 (5.0 %) | – | |
| Weight loss | N/A | – | |
| Present/absent | 390 (72.1 %)/112 (20.7 %) | – | |
| Missing | 39 (7.2 %) | – | |
| Chemotherapy | N/A | – | |
| Complete course | 59 (10.9 %) | – | |
| Incomplete course | 142 (26.3 %) | – | |
| No chemotherapy | 183 (33.8 %) | – | |
| Missing | 157 (29.0 %) | – | |
| Survival status | N/A | – | |
| Dead/living | 316 (58.4 %)/68 (12.6 %) | – | |
| Lost to follow-up | 157 (29.0 %) | – |
Table 2.
Genotype and allele frequency distribution of KRAS (rs61764370) gene polymorphism in gallbladder cancer patients and healthy controls
| Polymorphism | Cases (n = 541) | Control (n = 307) | Odds ratio (95 % confidence interval) | p value |
|---|---|---|---|---|
| rs61764370 | ||||
| Wild (TT) | 498 | 297 | 1 Ref | 1 Ref |
| Variant (heterozygous + mutant) (TG + GG) | 43 | 10 | 2.56 (1.27–5.18) | 0.007 |
| Allele carriage | ||||
| T allele | 1039 | 604 | 1 Ref | 1 Ref |
| G allele | 604 | 10 | 2.5 (1.25–5.01) | 0.008 |
Differences in KRAS-variant allele frequencies between cases and controls were tested for significance by a χ 2-test. Case–control odds ratios and their confidence intervals were calculated using Woolf approximations
p < 0.05 was considered as statistically significant
Till the last follow-up, only 68 (12.6 %) patients were found to be surviving, 316 patients were dead while 157 (29.0 %) were lost to follow-up. Kaplan–Meier survival curve indicates significant decrease (p < 0.0001) in overall survival for patients carrying heterozygous genotype (hazard ratio = 3.42, 95 % CI 1.21–4.20) (Fig. 1). Also, significant decrease in overall survival was observed for patient carrying allele G as compared with allele T (p < 0.0001, HR = 2.89, 95 % CI 1.21–4.20). We do not observe change in mean overall survival for any of the pathological parameters (stage of tumor, tumor invasion, node involvement, and distant metastasis) in respect with KRAS genotype (Table 3). Also, no difference in mean survival was observed for presence or absence of gallstones, jaundice, gallbladder mass, weight loss, and status of adjuvant chemotherapy clinical parameters (Table 4).
Fig. 1.
Kaplan–Meier overall survival curve of Gallbladder cancer patients (n = 541) for KRAS (rs61764370) gene polymorphism. Statistical analysis using the log-rank test indicates significant difference between the survival curves
Table 3.
Table showing association between various pathological characteristics with overall survival of GBC patients
| Tumor stage (n = 367) | Mean survival in months ± SD | p value | |
|---|---|---|---|
| Wild genotype (TT) (n = 297) | Heterozygous + mutant (TG + GG) (29) | ||
| 1a (n = 1) | 24.0 (n = 1) | – | – |
| 1b (n = 5) | 18.75 ± 7.89 (n = 4) | 24.0 (n = 1) | 0.349 |
| 2 (n = 19) | 14.53 ± 4.56 (n = 19) | – | – |
| 3a (n = 42) | 11.04 ± 8.0 (n = 40) | 4.39 ± 5.66 (n = 2) | 0.349 |
| 3b (n = 48) | 8.09 ± 3.1 (n = 46) | 6.05 ± 8.41 (n = 2) | 0.399 |
| 4a (n = 89) | 3.23 ± 2.24 (n = 76) | 2.44 ± 1.44 (n = 13) | 0.224 |
| 4b (n = 163) | 1.97 ± 1.18 (n = 141) | 1.48 ± 0.94 (n = 22) | 0.063 |
| T status (n = 349) | Mean survival in months ± SD | p value | |
|---|---|---|---|
| Wild genotype (TT) (n = 310) | Heterozygous + mutant (TG + GG) (n = 39) | ||
| T1 (n = 4) | 15.75 ± 9.53 (n = 4) | – | – |
| T2 (n = 67) | 7.84 ± 4.91 (n = 65) | 7.95 ± 5.73 (n = 2) | 0.976 |
| T3 (n = 115) | 5.85 ± 5.73 (n = 104) | 2.37 ± 3.42 (n = 11) | 0.051 |
| T4 (n = 163) | 3.54 ± 3.62 (n = 137) | 2.78 ± 4.49 (n = 26) | 0.345 |
| N status (n = 296) | Mean survival in months ± SD | p value | |
|---|---|---|---|
| Wild genotype (TT) (n = 266) | Heterozygous + mutant (TG + GG) (n = 30) | ||
| N0 (n = 85) | 9.91 ± 6.18 (n = 77) | 4.06 ± 3.43 (n = 8) | 0.01 |
| N1 (n = 102) | 4.86 ± 3.66 (n = 93) | 3.09 ± 3.52 (n = 9) | 0.166 |
| N2 (n = 109) | 2.62 ± 2.54 (n = 96) | 1.4 ± 1.04 (n = 13) | 0.092 |
| M status (n = 344) | Mean survival in months ± SD | p value | |
|---|---|---|---|
| Wild genotype (TT) (n = 306) | Heterozygous + mutant (TG + GG) (n = 38) | ||
| M0 (n = 217) | 6.95 ± 5.64 (n = 196) | 4.22 ± 5.55 (n = 21) | 0.04 |
| M1 (n = 127) | 2.51 ± 1.38 (n = 110) | 2.36 ± 0.81 (n = 17) | 0.05 |
Statistical analysis done using independent Student t test
p < 0.05 was considered as statistically significant
Table 4.
Table showing association of various clinicopathological parameters with overall survival of GBC patients
| Gallstone (n = 365) | Mean survival in months ± SD | p value | |
|---|---|---|---|
| Wild genotype (TT) | Heterozygous + mutant (TG + GG) | ||
| Present (n = 302) | 5.28 ± 5.19 (n = 269) | 2.69 ± 4.41 (n = 33) | 0.09 |
| Absent (n = 63) | 4.83 ± 4.83 (n = 54) | 3.27 ± 3.43 (n = 9) | 0.36 |
| Jaundice (n = 371) | Mean survival in months ± SD | p value | |
|---|---|---|---|
| Wild genotype (TT) (n = 331) | Heterozygous + mutant (TG + GG) (n = 40) | ||
| Present (n = 261) | 3.09 ± 2.96 (n = 228) | 1.78 ± 1.29 (n = 33) | 0.01 |
| Absent (n = 110) | 9.76 ± 5.79 (n = 103) | 9.76 ± 5.79 (n = 103) | 0.39 |
| Palpable gallbladder mass (n = 374) | Mean survival in months ± SD | p value | |
|---|---|---|---|
| Wild genotype (TT) (n = 334) | Heterozygous + mutant (TG + GG) (n = 40) | ||
| Present (n = 270) | 3.17 ± 2.8 (n = 234) | 2.04 ± 2.12 (n = 36) | 0.02 |
| Absent (n = 104) | 9.77 ± 6.15 (n = 100) | 9.87 ± 10.58 (n = 4) | 0.98 |
| Weight loss (n = 373) | Mean survival in months ± SD | p value | |
|---|---|---|---|
| Wild genotype (TT) (n = 332) | Heterozygous + mutant (TG + GG) (n = 41) | ||
| Present (n = 288) | 3.69 ± 3.53 (n = 250) | 1.83 ± 1.26 (n = 38) | 0.001 |
| Absent (n = 85) | 9.63 ± 6.39 (n = 82) | 16.00 ± 6.93 (n = 3) | 0.09 |
| Chemotherapy (n = 382) | Mean survival in months ± SD | p value | |
|---|---|---|---|
| Wild genotype (TT) (n = 340) | Heterozygous + mutant (TG + GG) (n = 42) | ||
| Complete (n = 59) | 14.35 ± 4.29 (n = 56) | 16.0 ± 6.93 (n = 3) | 0.53 |
| Incomplete (n = 142) | 5.42 ± 2.48 (n = 132) | 3.41 ± 0.81 (n = 10) | 0.01 |
| No chemotherapy (n = 181) | 1.49 ± 0.76 (n = 152) | 1.24 ± 0.84 (n = 29) | 0.11 |
Statistical analysis done using independent Student t test
p < 0.05 was considered as statistically significant
Discussion
To the best of our knowledge, this is the first study evaluating the association of rs61764370 polymorphism with susceptibility and prognosis of gallbladder malignancy in this endemic belt. The study results show that heterozygous genotype (TG) and mutant allele (G) is associated with increased risk of GBC with worse outcomes in North Indian population. Heterozygous carrier carries significant higher risk (odds ratio = 2.56, 95 % CI 1.27–5.18, p = 0.007) of GBC with respect to homozygous wild genotype (TT). Similarly, at allelic level, patients with allele G have higher risk of malignancy (odds ratio = 2.5, 95 % CI 1.25–5.01, p = 0.008) in comparison with allele T. Subsequent limitation of the current study is of sample size, and these findings require further validation. In our cohort of 838 subjects, we didn’t find mutant genotype (GG) and hence all comparison is done with reference to heterozygous genotype (TG). Our results are consistent with Cerne et al. (2012) who have made a similar observation in a large sample pool. Absence of detection of mutant genotype (GG) in this study may be due to its very low frequency in North Indian population. However, this hypothesis needs to be validated in further studies.
The association of KRAS rs61764370 polymorphism with susceptibility to head and neck, ovarian, oral, breast, colorectal and non-small cell lung cancer has been widely studied (Cipollini et al. 2014). Paranjape et al. (2011) identified variant genotype as a genetic marker for triple-negative breast cancer in premenopausal women. In addition, altered gene expression and lower level of let-7 miRNA has been observed for KRAS-variant tumors. Dai et al. (2015) observed a functional role of rs61764370 polymorphism in colorectal cancer development and oxaliplatin-based chemotherapy response. Significant association is also observed in females who develop both breast and ovarian cancer with uninformative BRCA sequencing results (Pilarski et al. 2012). In line with these results, we have also observed significant association of rs61764370 polymorphism with susceptibility of GBC. These findings indicate possible association of the variant genotype with the risk of malignancy. An account of this association may be lowered cellular levels of let-7 which may increase cell growth or altered let-7 binding in the 3′UTR which may lead to KRAS overexpression. However, Caiola et al. (2012) have not observed significant association of rs61764370 polymorphism with ovarian cancer. Also, in a large case control study, no significant association with ovarian or breast cancer has been observed (Ovarian Cancer Association Consortium, Breast Cancer Association Consortium, and Consortium of Modifiers of BRCA1 and BRCA2). These contradictory results may be due to population and patient selection bias and need further evaluation.
Most of the studies have revealed significant association of rs61764370 polymorphism with patient survival. Christensen et al. (2009) observed worse clinical outcome for oral cancer in relation to variant genotype. Similarly, patients with colorectal cancer with G allele (mutant) have a shorter survival and a higher risk of relapse or metastasis when compared to those carrying the wild allele (Dai et al. 2015). We also observed significant association of heterozygous genotype (TG) and mutant allele (G) with decreased overall survival of patients with gallbladder malignancy. In addition, survival of GBC patients in relation to rs61764370 polymorphism was found to be independent of any prognostic factor (stage of tumor, presence or absence of jaundice, gallbladder mass, chemotherapy, and weight loss). It is possible that a decrease in let-7 miRNA binding leads to a corresponding increase in KRAS protein expression which results in activated GTPase activity which in turn leads to poor prognosis in these patients. These results indicate that KRAS genotype (rs61764370) is an independent prognostic biomarker for gallbladder cancer for North Indian population.
In a study by Ganzinelli et al. (2015), no evidence of association was observed for overall survival and progression-free survival in non-small cell lung cancer in regards to this polymorphism. Also, better prognosis was observed for patients treated with docetaxel instead of erlotinib for both overall survival and progression-free survival. A study by Caiola et al. (2012) also has not demonstrated an association of rs61764370 polymorphism with outcome or physiopathological characteristic in ovarian cancer. These differences may be due to heterogeneity of the disease and impact of several confounding factors on the results.
We are now beginning to understand the importance of 3′ untranslated region miRNA binding site polymorphism in cancer biology (Pelletier and Weidhaas 2010). Better understanding of miRNA, miRNA binding site polymorphism along with its functional role will be very useful towards clinical utilization of these new genetic variations. Also, validation of these variants as a diagnostic, prognostic, and predictive biomarker for gallbladder cancer would provide a valuable clinical tool (Kim and Slack 2014).
To conclude, in our pilot study, we have demonstrated that KRAS rs61764370 polymorphism is significantly associated with risk and prognosis of gallbladder malignancy in this endemic belt. We hope that our results will help in characterizing these variants as potential biomarker of cancer outcome, treatment response, and disease management, heading towards personalized medicine.
Acknowledgments
The authors are thankful to the Council of Science and Technology, Government of Uttar Pradesh, Lucknow, India for financially supporting the study (Grant No. CST/SERPD/D-3429). Hasan Raza Kazmi is thankful to Indian Council of Medical Research, Government of India, New Delhi, India for providing fellowship (Grant No. 3/2/2/60/2011/NCDIII).
Funding
The study was funded by Council of Science and Technology, Government of Uttar Pradesh, India (Grant No. CST/SERPD/D-3429) and Indian Council of Medical Research, Government of India, India (Grant No. 3/2/2/60/2011/NCDIII).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Human and animals rights
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Informed consent
Informed consent was obtained from all individual participants included in the study.
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