Genetic Mutations Associated With Cigarette Smoking in Pancreatic Cancer
Amanda Blackford
Giovanni Parmigiani
Thomas W Kensler
Christopher Wolfgang
Siân Jones
Xiaosong Zhang
D Willams Parsons
Jimmy Cheng-Ho Lin
Rebecca J Leary
James R Eshleman
Michael Goggins
Elizabeth M Jaffee
Christine A Iacobuzio-Donahue
Anirban Maitra
Alison Klein
John L Cameron
Kelly Olino
Richard Schulick
Jordan Winter
Bert Vogelstein
Victor E Velculescu
Kenneth W Kinzler
Ralph H Hruban
Corresponding author: Ralph H. Hruban, M.D., The Johns Hopkins Medical Institutions, Department of Pathology, Weinberg 2249, 401 North Broadway, Baltimore, Maryland 21231, Phone: (410)955-9132, Email:rhruban@jhmi.edu, Office Fax: (410)955-0115
Issue date 2009 Apr 15.
Abstract
Background
Cigarette smoking doubles the risk of pancreatic cancer and smoking accounts for 20 to 25% of pancreatic cancers. The recent sequencing of the pancreatic cancer genome provides an unprecedented opportunity to identify mutational patterns associated with smoking.
Design
We previously sequenced over 750 million base pairs of DNA from 23,219 transcripts in 24 adenocarcinomas of the pancreas (“Discovery Screen”). In this previous study the 39 genes that were mutated more than once in the Discovery Screen were sequenced in an additional 90 adenocarcinomas of the pancreas (“Validation Screen”). Here we compared the somatic mutations in the cancers obtained from individuals who ever smoked cigarettes (n=64) to the somatic mutations in the cancers obtained from individuals who never smoked cigarettes (n=50).
Results
When adjusted for age and gender, analyses of the Discovery Screen revealed significantly more non-synonymous mutations in the carcinomas obtained from ever smokers (mean 53.1 mutations per tumor, SD 27.9) than in the carcinomas obtained from never smokers (mean 38.5, SD 11.1, p=0.04). The difference between smokers and non-smokers was not driven by mutations in known driver genes in pancreatic cancer (KRAS,TP53,p16/CDKN2A andSMAD4), but instead was predominantly observed in genes mutated at lower frequency. No differences were observed in mutations in carcinomas from the head vs. tail of the gland.
Conclusion
Pancreatic carcinomas from cigarette smokers harbor more mutations than do carcinomas from never smokers. The types and patterns of these mutations provide insight into the mechanisms by which cigarette smoking causes pancreatic cancer.
INTRODUCTION
Pancreatic cancer is the fourth leading cause of cancer death in the United States [1]. It has been estimated that in the year 2008, approximately 37,680 Americans were diagnosed with pancreatic cancer, and that 34,290 died from this disease [1]. A number of factors have been identified that increase the risk of pancreatic cancer, including advancing age, diets high in meats and fats, diets low in vegetables and folate, diabetes mellitus, obesity, chronic pancreatitis, partial gastrectomy, radiation, a family history of pancreatic cancer, and cigarette smoking [2–6]. Of all of these known risk factors, cigarette smoking remains the leading preventable cause of pancreatic cancer [6,7]. Approximately 20% of cancers of the pancreas are caused by cigarette smoking, and a recent meta-analysis of 82 studies published between 1950 and 2007 on smoking and pancreatic cancer found that current smokers have a 1.74 fold (95% CI 1.61–1.87) increased risk of developing pancreatic cancer [6,7]. Smoking has also been associated with early onset pancreatic cancer and smoking cessation has been shown to reduce pancreatic cancer risk [8–12].
Genetic analyses of other cancers caused by cigarette smoking have revealed increased numbers of mutations in cancer-associated genes as well as specific types of mutations in cancers resected from smokers [13–19]. This link between cigarette smoking and specific genetic changes in a cancer is strongest for lung carcinomas [14,19]. Smoking is associated with an approximate 11-fold increased relative risk of lung cancer, and activating point mutations in theKRAS gene are more common in adenocarcinomas of the lung resected from smokers than they are in adenocarcinomas from non-smokers [16,19–21] Most of these mutations are G:C to T:A transversions, a mutation type associated with carcinogens such as polycyclic aromatic hydrocarbons in tobacco smoke [16,22]. Remarkably, these same mutations can be seen in lung cancers obtained from ex-smokers, suggesting that theseKRAS gene mutations occurred years before the cancers were resected [15]. Similarly, a number of studies have shown thatTP53 gene mutations are more common in lung cancers from smokers than they are in lung cancers from never smokers, and, again, the G:C to T:A transversions predominate with a specificity towards CpG sites [14,17,23–25]. Thus, there is a strong “fingerprint” of tobacco carcinogens in the DNA of lung cancer [24,26].
The recent analysis of the “pancreatic cancer genome,” encompassing the sequencing of 20,661 protein coding genes in a series of 24 pancreatic cancers, provides a unique opportunity to correlate the somatic genetic changes in pancreatic cancer with smoking status [27]. In this previous study over 750 million base pairs of DNA were sequenced in two phases [27]. First, in the “Discovery Screen,” the sequences of the protein-coding exons from 20,661 genes were sequenced in 24 advanced adenocarcinomas of the pancreas. Of the 1562 somatic mutations discovered using this approach, 62.4% were missense, 25.5% were synonymous, 5.0% were small insertions or deletions, 3.8% were nonsense, and 3.3% were in splice sites or within untranslated regions (UTR) [27]. In addition, 198 homozygous deletions and 144 high copy number amplifications were identified in the cancers included in the Discovery Screen using high density oligonucleotide arrays [27]. In the second phase of this study, the “Validation Screen”, 39 genes that were mutated more than once in the Discovery Screen were sequenced in an additional panel of 90 well-characterized adenocarcinomas of the pancreas [27].
Here we correlate these data with patient smoking history as well as with a variety of other clinical factors such as patient age, sex, stage, and location of the cancer within the pancreas.
MATERIALS AND METHODS
This study was approved by the Johns Hopkins Institutional Review Board.
Patients
All available records were retrospectively reviewed on the 114 patients (24 in the Discovery Screen and 90 from the Validation Screen). This included a review of the patient’s hospital charts, the electronic patient medical records, and the Johns Hopkins Pancreatic Cancer Research Database [28]. Ninety-eight (86%) of the 114 patients included in this study were deceased at the time of the study.
Non-smokers were defined as patients who reported that they had never smoked in their lives. Smokers were defined as patients who reported that they had smoked in their lives. Ex-smokers were defined as smokers who had quit more than one year prior to surgery for their pancreatic cancer. Information was not available on second-hand smoking exposure.
Statistical analyses
The total numbers of mutations, deletions and amplifications were compared between clinical parameters using a Poisson regression model that adjusted for smoking status and included an over-dispersion term to account for patient-to-patient variation. A similar approach was used to compare the number of mutations between smokers and non-smokers, adjusting for age and gender. Analyses were adjusted for gender because genes specific to the Y chromosome were not sequenced and therefore more alleles were sequenced in the cancers obtained from women then in the cancers obtained from men [27]. The difference in frequency of specific mutation types (base pair changes and insertions/deletions) and the context in which the mutations occurred, were compared between smokers and non-smokers using mixed-effect logistic regression models that adjusted for age and gender. The difference in frequency of mutations and deletions of the known driver genes (i.e.KRAS,TP53,SMAD4 andCDKN2A/p16) between smokers and non-smokers was evaluated with Fisher’s Exact test. The number of statistical comparisons was not defined prior to the analyses, therefore the p values presented are not adjusted for the number of comparisons and are included for descriptive purposes only.
RESULTS
Patient demographics
A summary of the patient demographics for the Discovery and Validation Screens is provided inTable 1, and the smoking histories of each of the patients included in the original sequencing study are provided inSupplementary Tables S1 andS2[27]. Briefly, the mean age for both smokers and non-smokers was 65 years. The Discovery Screen included 10 males and 14 females, and the Validation Screen 43 males and 47 females[27]. Sixty-four of the 114 patients included were smokers, and 50 were non-smokers. Of the 64 smokers, 38 had reported that they had quit smoking, and 26 of the 38 ex-smokers had quit more than ten years before their diagnosis. The smokers in the Discovery Screen smoked a mean of 43 pack-years, and the smokers in the Validation Screen a mean of 38 pack-years. There were no p-values of 0.05 or less for any of the clinical parameters examined between the smokers and non-smokers (Table 1).
Table 1.
Distribution of clinical and smoking characteristics among smokers and non-smokers, separately for the Discovery and Validation screens.
| Discovery Screen | Prevalence Screen | |||
|---|---|---|---|---|
| Non-smokers N = 13 | Smokers N = 11 | Non-smokers N = 37 | Smokers N = 53 | |
| Age – Mean (SD) | 64.7 (12.1) | 65.5 (9.7) | 64.9 (11.3) | 65.2 (9.6) |
| Gender – No. (%) | ||||
| Male | 6 (46.2) | 4 (36.4) | 22 (59.5) | 21 (39.6) |
| Female | 7 (53.8) | 7 (63.6) | 15 (40.5) | 32 (60.4) |
| Race – No. (%) | ||||
| White | 12 (92.3) | 9 (81.8) | 32 (86.5) | 47 (88.7) |
| Other Race | 1 (7.7) | 2 (18.2) | 5 (13.5) | 6 (11.3) |
| Surgery – No. (%) | ||||
| Autopsy | 4 (30.8) | 3 (27.3) | 1 (2.7) | 0 (0.0) |
| Whipple | 9 (69.2) | 6 (54.5) | 33 (89.2) | 43 (81.1) |
| Distal Pancreatectomy | 0 (0.0) | 2 (18.2) | 3 (8.1) | 10 (18.9) |
| Location of Tumor – No. (%) | ||||
| Head | 12 (92.3) | 7 (63.6) | 34 (94.4) | 43 (84.3) |
| Tail | 1 (7.7) | 4 (36.4) | 2 (5.6) | 8 (15.7 |
| Grade – No. (%) | ||||
| Poor | 8 (61.5) | 7 (70) | 11 (29.7) | 22 (42.3) |
| Moderate/Well | 5 (38.5) | 3 (30) | 26 (70.3) | 30 (57.7) |
| Tumor Size – No. (%) | ||||
| < 3 cm | 3 (23.1) | 2 (18.2) | 13 (35.1) | 19 (35.8) |
| 3–5 cm | 6 (46.1) | 6 (54.5) | 20 (54.1) | 25 (47.2) |
| > 5 cm | 2 (15.4) | 2 (18.2) | 3 (8.1) | 8 (15.1) |
| Unknown (autopsy cases) | 2 (15.4) | 1 (9.1) | 1 (2.7) | 1 (1.9) |
| Margin – No. (%) | ||||
| Negative | 6 (46.2) | 5 (45.5) | 23 (62.2) | 37 (69.8) |
| Positive | 4 (30.8) | 3 (27.3) | 13 (35.1) | 16 (30.2) |
| Unknown (autopsy cases) | 3 (23.1) | 3 (27.3) | 1 (2.7) | 0 (0.0) |
| Diabetic – No. (%) | ||||
| No | 9 (69.2) | 7 (63.6) | 7 (87.5) | 4 (50) |
| Yes | 4 (30.8) | 4 (36.4) | 1 (12.5) | 4 (50) |
| No. of Positive Lymph Nodes – Mean (SD) | 3 (1.7) | 6 (3.6) | 3 (4.6) | 3 (3) |
| No. of Lymph Nodes – Mean (SD) | 22 (12.9) | 18 (5.3) | 15 (8.2) | 15 (8.2) |
| Smoking Status – No. (%) | ||||
| Current | 5 (45.5) | 21 (39.6) | ||
| Former | 6 (54.5) | 32 (60.4) | ||
| Years Quit – No. (%) | ||||
| ≤ 10 | 2 (33.3) | 10 (31.2) | ||
| > 10 | 4 (66.7) | 22 (68.8) | ||
| Pack-Years – Mean (SD) | 43 (23.3) | 38 (27.9) | ||
Mutations in the Discovery Screen
We first examined the mutations identified by sequencing in the Discovery Screen and calculated the total number of mutations per sample for each of the clinical parameters evaluated (Table 2).
Table 2.
Mean (SD) genetic alterations for the Discovery Screen, by clinical parameters. P values for smoking-adjusted differences in the rate of mutations between patient groups. Differences between males and females exclude mutations on chromosome X. Values for age in years are regression coefficients (standard errors) for the average increase in the number of alterations with a yearly increase in age, adjusting for smoking status.
| N | Mutations | Deletions | Amplifications | Mutations, Deletions & Amplifications | |||||
|---|---|---|---|---|---|---|---|---|---|
| Mean (SD) | P | Mean (SD) | P | Mean (SD) | P | Mean (SD) | P | ||
| Nonsmoker | 13 | 56.2 (13.9) | 0.08 | 7.8 (4.7) | 0.58 | 5.5 (5.4) | 0.73 | 69.5 (16.4) | 0.08 |
| Smoker | 11 | 75.5 (41.7) | 8.8 (4.3) | 6.5 (9.1) | 90.9 (44.4) | ||||
| Moderate/Well grade | 8 | 58.1 (15.6) | 0.59 | 6.5 (2.7) | 0.23 | 2.6 (4.6) | 0.06 | 67.2 (19) | 0.28 |
| Poor Grade | 15 | 68.9 (37.4) | 8.7 (4.8) | 7.4 (7.9) | 84.9 (39) | ||||
| Aged < 70 | 16 | 60.8 (19.1) | 0.16 | 8.9 (4.6) | 0.33 | 6.6 (7.3) | 0.59 | 76.2 (24.3) | .37 |
| Aged ≥ 70 | 8 | 73.8 (47.1) | 7 (4.2) | 4.9 (7.3) | 85.6 (48.2) | ||||
| Black | 3 | 61.3 (15.9) | 0.69 | 7 (2) | 0.54 | 0.3 (0.6) | 0.01 | 68.7 (17.2) | 0.39 |
| White | 21 | 65.6 (32.7) | 8.4 (4.7) | 6.8 (7.3) | 80.9 (35.1) | ||||
| Female | 10 | 54.3 (13.5) | 0.35 | 6.4 (3.6) | 0.08 | 3 (4.4) | 0.05 | 63.7 (14.5) | 0.10 |
| Male | 14 | 69.3 (37) | 9.6 (4.7) | 8.1 (8.1) | 87 (38.7) | ||||
| Non-Diabetic | 16 | 68.9 (36) | 0.34 | 8.9 (5.1) | 0.32 | 7.8 (7.9) | 0.04 | 85.6 (37.4) | 0.13 |
| Diabetic | 8 | 57.4 (15.9) | 7 (2.7) | 2.4 (3.9) | 66.8 (19.8) | ||||
| Tail | 5 | 72.6 (24) | 0.88 | 7.8 (3.1) | 0.68 | 9 (10.9) | 0.39 | 89.4 (34.1) | 0.76 |
| Head | 19 | 63.1 (32.7) | 8.4 (4.8) | 5.2 (6) | 76.7 (33.6) | ||||
| Age in years | 0.96 (0.55) | 0.10 | −0.12 (0.08) | 0.16 | −0.06 (0.14) | 0.68 | 0.77 (0.61) | 0.22 | |
There was a trend for more mutations in smokers than in non-smokers. The number of mutations ranged from 40 to 187 per tumor for smokers and from 34 to 72 per tumor for non-smokers. As has been previously reported with lung cancer, the variance of the number of point mutations for smokers was higher than for non-smokers (Variance Ratio estimate = 9.0 (95% C.I. 2.7 – 32.7), p < 0.001) [19]. The eleven smokers had a mean of 75.5 intragenic mutations per carcinoma (SD 41.7) and the non-smokers a mean of 56.2 mutations (SD 13.9, p=0.06 when adjusted for age and gender) (Table 3). Thus, approximately 25% of the intragenic mutations in the pancreatic cancers obtained from smokers appear to be smoking-related.
Table 3.
Types of mutations by smoking status for the Discovery screen. P values for differences between smokers and non-smokers, adjusting for age and gender.
| Non-smokers N = 13 | Smokers N = 11 | P | |
|---|---|---|---|
| Mean SD | Mean SD | ||
| Mutations | 56.2 (13.9) | 75.5 (41.7) | 0.06 |
| Mutations not inKRAS, TP53,SMAD4 orCDKN2A/P16 | 53.9 (14.1) | 73.5 (41.7) | 0.05 |
| Mutations not in any driver gene | 53.2 (13.8) | 73.3 (41.8) | 0.05 |
| Synonymous Mutations | 14.8 (5.4) | 18.7 (11.3) | 0.26 |
| Non-synonymous Mutations | 38.5 (11.1) | 53.1 (27.9) | 0.04 |
| Transition Mutations | 35.3 (9) | 43.7 (16.9) | 0.04 |
| Transversion Mutations | 20.9 (10.3) | 31.8 (25.9) | 0.16 |
When homozygous deletions and amplifications were also included together with the mutations identified by sequencing, the carcinomas from the eleven smokers had a mean of 90.9 (SD 44.4) genetic alterations per tumor and the carcinomas from the non-smokers a mean of 69.5 (SD 16.4, p=0.08). There were no significant differences observed in the number of amplifications or in the number of deletions in smokers and non-smokers. Though the numbers were small, no significant differences were observed between the ex-smokers and the current smokers with respect to mutation number or type.
No significant differences were observed in the number of mutations for the other clinical variables examined for patients included in the Discovery Screen (Table 2).
Categories of Mutations in the Discovery Screen (Table 3)
Next we examined the broad categories of alterations observed in the Discovery Screen (Table 3). As noted above, the number of homozygous deletions and amplifications did not differ between the non-smokers and smokers. Our further analyses therefore focused on the mutations identified by sequencing.
When theKRAS andTP53 genes, the two previously reported targets of tobacco-related carcinogens, were excluded from the analyses a larger number of mutations were still identified in the cancers obtained from smokers (mean 73.9, SD 41.9) than in the cancers obtained from non-smokers (mean 54.4, SD 14.1, p=0.06 when adjusted for age and gender). A similar pattern was observed when all four “gene mountains,” theKRAS,TP53,SMAD4 andCDKN2A genes, were excluded from the analyses, with a mean of 73.5 (SD 41.7) mutations in the smokers and 53.9 (SD 14.1) mutations in the non-smokers (p=0.05 when adjusted for age and gender,Table 3) [27]. Finally, we compiled a list of 65 driver genes (Table S3). These 65 driver genes included genes identified in our previous genome-wide sequencing analyses, genes reported as driver genes in the literature, and genes with greater than 10 alterations in the Cosmic database (December 20, 2008;http://www.sanger.ac.uk/genetics/CGP/cosmic/) [27,29–31]. When these driver genes were excluded from the analyses, the difference persisted, with a mean of 73.3 (SD 41.8) mutations in smokers and 53.2 (SD 13.8) mutations in the non-smokers (p=0.05 when adjusted for age and gender). These results suggest that the differences observed in the number of mutations detected by sequencing between smokers and non-smokers are not driven by these major driver genes.
Significantly more non-synonymous mutations were observed in the cancers from smokers (mean 53.1, SD 27.9) than in the cancers from non-smokers (mean 38.5, SD 11.1, p=0.04 when adjusted for age and gender). More synonymous mutations were also observed in the cancers from smokers (mean 18.7, SD 11.3,Table 3) as compared to non-smokers (mean 14.8, SD 5.4, p=0.26), but this difference was not statistically significant.
Transitions were more common in the cancers from smokers (mean 43.7, SD 16.9) than in the cancers from non-smokers (mean 35.3, SD 9, p=0.04 when adjusted for age and gender). There were also more transversions in the cancers from smokers (mean 31.8, SD 25.9) than non-smokers (mean 20.9, SD 10.3), but this latter difference was not statistically significant (Table 3).
Types of Mutations in the Discovery Screen (Table 4)
Table 4.
Frequency of specific sequence mutations and context for the Discovery Screen, by smoking status. Odds ratios for having each specific type of mutation for smokers v. non-smokers, adjusting for age and gender.
| Number of mutations in non-smokers | Number of non-smokers with a specific mutation | Number of mutations in smokers | Number of smokers with a specific mutation | Odds Ratio [95% C.I.] | P | |
|---|---|---|---|---|---|---|
| Sequence Mutations | ||||||
| A to C | 26 | 10 | 22 | 10 | 0.72 [0.33,1.54] | 0.40 |
| A to G | 32 | 12 | 50 | 11 | 1.38 [0.86,2.21] | 0.18 |
| A to T | 20 | 9 | 29 | 11 | 1.25 [0.69,2.27] | 0.46 |
| C to A | 36 | 13 | 70 | 10 | 1.6 [1.04,2.46] | 0.03 |
| C to G | 34 | 11 | 34 | 10 | 0.76 [0.45,1.26] | 0.28 |
| C to T | 214 | 13 | 197 | 11 | 0.78 [0.57,1.07] | 0.13 |
| G to A | 187 | 13 | 200 | 11 | 1.02 [0.79,1.31] | 0.89 |
| G to C | 39 | 9 | 35 | 10 | 0.8 [0.36,1.76] | 0.57 |
| G to T | 57 | 13 | 83 | 11 | 1.27 [0.88,1.85] | 0.20 |
| T to A | 8 | 6 | 20 | 8 | 2.32 [0.99,5.45] | 0.05 |
| T to C | 26 | 11 | 34 | 8 | 1.15 [0.64,2.06] | 0.63 |
| T to G | 15 | 8 | 16 | 7 | 0.85 [0.41,1.79] | 0.68 |
| Insertion or Deletion | 37 | 13 | 41 | 11 | 1.01 [0.63,1.63] | 0.96 |
| Context | ||||||
| A | 78 | 13 | 101 | 11 | 1.07 [0.78,1.48] | 0.66 |
| C | 90 | 13 | 104 | 11 | 0.91 [0.67,1.25] | 0.57 |
| C*pG | 152 | 13 | 140 | 11 | 0.82 [0.6,1.12] | 0.21 |
| CpG* | 128 | 13 | 143 | 11 | 1.16 [0.8,1.69] | 0.43 |
| G | 92 | 13 | 119 | 11 | 1.11 [0.82,1.5] | 0.49 |
| G*pA | 63 | 12 | 56 | 11 | 0.79 [0.41,1.53] | 0.49 |
| T | 49 | 12 | 70 | 11 | 1.25 [0.81,1.92] | 0.31 |
| TpC* | 42 | 12 | 57 | 10 | 1.16 [0.76,1.77] | 0.49 |
| All Sequence Mutations | 731 | 13 | 831 | 11 | 1.26 [0.74,2.15] | 0.40 |
We next examined the specific types of mutations identified in the Discovery Screen (Table 4). Here the mutations were placed into one of thirteen groups: the 12 possible base pair changes (based on the reading strand) and insertions or deletions. Of the 13 possible mutation types, C:G to A:T (Odds Ratio 1.6, 95% CI [1.04,2.46], p=0.03 adjusted age and gender) and T:A to A:T mutations (Odds Ratio 2.32, 95% CI [0.99,5.45], p=0.05 adjusted age and gender) were both more common in the cancers from smokers than in the cancers from non-smokers.
There were no significant differences observed in the context in which the mutations occurred (Table 4). Similar analyses for the Validation Screen are presented inTable S4.
TP53 gene mutations and smoking
Point mutations in theTP53 gene were identified in 82% of the cancers (Table 5). Eighteen of the 24 cancers in the Discovery Screen harbored aTP53 gene mutation, as did 76 of the 90 cancers in the Validation Screen. The prevalence ofTP53 gene mutations in cancers from smokers did not differ significantly from the prevalence ofTP53 gene mutations in the cancers from non-smokers. Fifty (78%) of the 64 cancers from smokers harbored aTP53 gene mutation, compared to 44 (88%) of the 50 cancers from non-smokers (p=0.22). In addition, the types and context of theTP53 gene mutations in smokers and in non-smokers also were similar (Table 6).
Table 5.
Frequency distribution of the number of patients with none or at least one sequencing mutation inKRAS, SMAD4, CDKN2A/P16 orTP53. (NS= non-smoker, S= smoker)
| Discovery Screen | Validation Screen | Combined Samples | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Non-smokers | Smokers | P | Non-smokers | Smokers | P | Non-smokers | Smokers | P | |||||||
| N | % | N | % | N | % | N | % | N | % | N | % | ||||
| KRAS | |||||||||||||||
| Wild-type | 0 | (0) | 0 | (0) | - | 0 | (0) | 1 | (2) | - | 0 | (0) | 1 | (2) | 1.00 |
| Mutated | 13 | (100) | 11 | (100) | 37 | (100) | 52 | (98) | 50 | (100) | 63 | (98) | |||
| SMAD4 | |||||||||||||||
| Wild-type | 8 | (62) | 8 | (73) | 0.68 | 28 | (76) | 39 | (74) | 1.00 | 36 | (72) | 47 | (73) | 1.00 |
| Mutated | 5 | (38) | 3 | (27) | 9 | (24) | 14 | (26) | 14 | (28) | 17 | (27) | |||
| CDKN2A/p16 | |||||||||||||||
| Wild-type | 12 | (92) | 10 | (91) | 1.00 | 30 | (81) | 38 | (72) | 0.33 | 42 | (84) | 48 | (75) | 0.26 |
| Mutated | 1 | (8) | 1 | (9) | 7 | (19) | 15 | (28) | 8 | (16) | 16 | (25) | |||
| TP53 | |||||||||||||||
| Wild-type | 2 | (15) | 4 | (36) | 0.36 | 4 | (11) | 10 | (19) | 0.38 | 6 | (12) | 14 | (22) | 0.22 |
| Mutated | 11 | (85) | 7 | (64) | 33 | (89) | 43 | (81) | 44 | (88) | 50 | (78) | |||
Table 6.
Frequency distribution of the number of sequencing mutations inKRAS, SMAD4, TP53, CDKN2A/p16 and all other genes, by mutation type and context, comparing non-smokers and smokers, adjusting for age and gender. Some patients had more than one mutation on an individual gene, and total represents the number of mutations across all patients within smoking category (NS= non-smoker, S= smoker).
| KRAS | SMAD4 | TP53 | CDKN2A/p16 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| NS | S | P | NS | S | P | NS | S | P | NS | S | P | ||
| Sequence Mutations | |||||||||||||
| A to C | 1 | 2 | 0.99 | 1 | 0 | >0.99 | 0 | 1 | >0.99 | 0 | 1 | >0.99 | |
| A to G | 0 | 1 | >0.99 | 0 | 2 | >0.99 | 4 | 3 | 0.89 | 0 | 0 | - | |
| A to T | 0 | 0 | - | 0 | 0 | - | 2 | 1 | 0.99 | 0 | 1 | >0.99 | |
| C to A | 0 | 0 | - | 0 | 0 | - | 1 | 2 | 0.99 | 0 | 1 | >0.99 | |
| C to G | 0 | 0 | - | 1 | 0 | >0.99 | 1 | 2 | 0.99 | 1 | 2 | 0.85 | |
| C to T | 0 | 0 | - | 6 | 3 | 0.17 | 9 | 10 | >0.99 | 4 | 6 | 0.32 | |
| G to A | 26 | 34 | 0.9 | 0 | 3 | >0.99 | 9 | 12 | 0.58 | 0 | 2 | >0.99 | |
| G to C | 6 | 10 | 0.99 | 0 | 1 | >0.99 | 1 | 0 | >0.99 | 0 | 1 | >0.99 | |
| G to T | 17 | 17 | 0.37 | 2 | 1 | 0.91 | 5 | 4 | 0.85 | 1 | 1 | 0.99 | |
| InDel | 0 | 0 | - | 4 | 7 | 0.39 | 11 | 8 | 0.36 | 2 | 4 | 0.77 | |
| T to A | 0 | 0 | - | 0 | 0 | >0.99 | 1 | 2 | 0.99 | 0 | 0 | - | |
| T to C | 0 | 0 | - | 1 | 0 | >0.99 | 2 | 3 | 0.99 | 0 | 0 | - | |
| T to G | 0 | 0 | - | 0 | 0 | - | 1 | 2 | 0.99 | 0 | 1 | >0.99 | |
| Context | |||||||||||||
| A | 1 | 3 | 0.99 | 1 | 2 | 0.99 | 6 | 5 | 0.86 | 0 | 2 | >0.99 | |
| C | 0 | 0 | - | 1 | 0 | >0.99 | 2 | 2 | >0.99 | 2 | 5 | >0.99 | |
| C*pG | 0 | 0 | - | 4 | 1 | 0.14 | 8 | 11 | 0.99 | 3 | 4 | 0.34 | |
| CpG* | 0 | 0 | - | 0 | 0 | - | 5 | 8 | 0.97 | 0 | 0 | - | |
| G | 49 | 61 | 0.99 | 1 | 4 | 0.98 | 4 | 7 | 0.99 | 0 | 3 | >0.99 | |
| G*pA | 0 | 0 | - | 1 | 1 | 0.99 | 6 | 1 | 0.67 | 1 | 1 | 0.99 | |
| T | 0 | 0 | - | 1 | 0 | >0.99 | 4 | 7 | 0.99 | 0 | 1 | >0.99 | |
| TpC* | 0 | 0 | - | 2 | 2 | >0.99 | 1 | 1 | >0.99 | 0 | 0 | ||
| Total Mutations | 50 | 64 | >0.99 | 15 | 17 | 0.12 | 47 | 50 | >0.99 | 8 | 20 | 0.02 | |
KRAS gene mutations and smoking
KRAS gene mutations were observed in 113 (99%) of the 114 pancreatic cancers sequenced. With almost universalKRAS gene mutations, the number ofKRAS gene mutations in the cancers from smokers did not differ significantly from the number in cancers from non-smokers. As was true for theTP53 gene, the types and context of theKRAS gene mutations in smokers and in non-smokers were similar (Table 6).
Other gene mutations and smoking
There were a total of 1562 sequence mutations involving 1315 unique genes in the tumor samples. In addition to the genes presented inTable 5,TTN was mutated in 8 carcinomas: 4 smokers and 4 non-smokers. Of the remaining 1310 genes, 1166 were mutated in only 1 tumor sample. The remaining 144 genes were mutated in 2, 3, or 4 tumor samples and were not analyzed for differences by smoking group.
DISCUSSION
A number of studies have linked cigarette smoking with specific genetic alterations in cancer-associated genes in lung cancer [14,16,17,19,20,23,24] For example, Westra et al. reported significantly moreKRAS gene mutations in lung adenocarcinomas obtained from current smokers (30%) and former smokers (32%) than in lung adenocarcinomas obtained from never smokers (7%, p=0.015) [15]. Similarly, Le Calvez and colleagues foundTP53 gene mutations in the lung cancers of 47.5% of never smokers, 55.6% of former smokers, and 77.4% of current smokers [14]. More recently, Ding et al. sequenced 623 genes in 188 adenocarcinomas of the lung and found significantly more mutations in the cancers from smokers than in the cancers from never smokers (p=0.02) [19]. All of the cancers obtained from never smokers harbored 5 or fewer mutations, while the cancers obtained from smokers had as many as 49 mutations [19]. Comparable results have been reported for other cancer types associated with cigarette smoking such as head and neck cancer, and bladder cancer [18,32,33]
Smoking also has been associated with pancreatic cancer through epidemiologic studies and smoking has been linked to specific genetic mutations in pancreatic cancers [6,13,34]. Pancreatic cancers from cigarette smokers have been reported to have moreKRAS and moreTP53 gene mutations than pancreatic cancers from non-smokers [13,34]. For example, Jiao et al. found that smoking was associated with G:C to A:T mutations in theKRAS gene in pancreatic cancer [35]. It should be noted, however, that not all studies have found a link between smoking and specific genetic changes in pancreatic cancer [36,37]. For example, Porta et al. reported on 107 pancreatic cancers and found no relationship betweenKRAS gene mutations and smoking [37].
The sequencing of the pancreatic cancer genome provided a unique opportunity to correlate cigarette smoking and other clinical parameters with specific genetic mutations [27]. We found that although the number of smoking related mutations did not appear to be as high as it was for lung cancer, pancreatic cancers obtained from ever smokers harbored more mutations than cancers obtained from never smokers [19]. As has been previously reported with lung cancer, the variance of the number of point mutations in the pancreatic cancers obtained from smokers was higher than the variance of the number of point mutations in the pancreatic cancers obtained from non-smokers[19]. We estimate that one in four of the mutations in the pancreatic cancers obtained from smokers may be smoking related.
In contrast to several previous reports, however, we did not observe an association between smoking andKRAS gene mutations [13]. This likely reflects the selection criteria used to include cases in the sequencing project [27,34,38]. Cancers with variant morphologies, such as medullary carcinoma, were excluded from the project in an effort to increase the uniformity of the cancers sequenced. Medullary carcinomas, as we have reported before, are often microsatellite unstable, they lackKRAS gene mutations, and some are caused by germline mutations in a DNA mismatch repair gene [39]. Thus, the selection criteria for the pancreatic cancer genome project tended to exclude theKRAS wild-type cases driven by a pathway unrelated to smoking. Simply put, with 99% of the cancers harboring aKRAS gene mutation, it would have been virtually impossible to detect an impact of smoking on theKRAS gene.
The differences between the number of mutations in smokers and non-smokers were not found in the other genes known to be “driver” genes in pancreatic cancer, such asTP53,CDKN2A andSMAD4 [27,40–44]. This observation can be explained by the fact that these mutations are likely required for pancreatic cancer to occur and are highly selected for during the tumorigenic process. Passenger mutations, but not driver mutations, provide a molecular clock that can be used to infer mutation rates [45]. Smokers may develop pancreatic cancer more frequently and at a younger age of onset, but the driver genes that are mutated appear to be the same in the two groups [8]. While this distinction between the passenger mutations and driver mutations has been overlooked in prior literature, it may explain the often unconvincing associations between smoking and driver genetic mutations[37].
We also examined the types of mutations and the context in which these mutations occurred. We did not identify a signature tobacco-related mutation in the smokers. A possible explanation for this heterogeneity is that it reflects the multiple DNA damaging compounds (>100) found in cigarette smoke, and that perhaps the mutagenicity of cigarette smoke is not limited to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP), two well-studied tobacco-derived carcinogens [46]. The data would also be consistent with the hypothesis that the carcinogens in tobacco damage the DNA in the pancreas in a non-specific way, but this latter hypothesis is not consistent with extensive data from the study of lung cancer, and with the finding of specific tobacco-derived carcinogens in the pancreatic juice of smokers. Other possible explanations include that non-tobacco-related mutagenic risk factors for pancreatic cancer may share mutagenic properties with the tobacco mutagens active in pancreatic tissues, and that the end-organ metabolic products of diverse tobacco carcinogens differ in the lung and the pancreas[19,47].
We examined the number of mutations in the cancers relative to a number of other clinical parameters, such as location within the pancreas (head vs. tail), sex of the patient, age of the patient, tumor grade, margin status, and stage. No statistically significant differences were found.
Limitations of this study should be acknowledged. Because 86% of the patients were deceased at the time of this study, all of the clinical parameters were collected retrospectively by review of the patient’s hospital charts, the electronic patient medical records, and the Johns Hopkins Pancreatic Cancer Research Database [28]. While several studies have suggested that self-reporting may under estimate cigarette smoking, the magnitude of this under-reporting is likely small enough to have only a modest impact on our results [48–50].
In conclusion, we found that cigarette smoking is associated with greater numbers of mutations in pancreatic cancer, but that these mutations do not produce a characteristic profile.
Supplementary Material
Acknowledgments
The authors thank Dr. Scott Kern for his helpful suggestions.
Supported by the NIH SPORE (Specialized Programs of Research Excellence) in Gastrointestinal Cancer Grant CA62924, The Sol Goldman Pancreatic Cancer Research Center, The Lustgarten Foundation for Pancreatic Cancer Research, and the Michael Rolfe Pancreatic Cancer Foundation
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