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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: Environ Mol Mutagen. 2019 May 23;60(8):693–703. doi: 10.1002/em.22296

Concentrations of trace elements and KRAS mutations in pancreatic ductal adenocarcinoma

Álvaro Gómez-Tomás 1,2, José Pumarega 3,4, Juan Alguacil 3,5, André FS Amaral 6, Núria Malats 7,8, Natàlia Pallarès 1,4, Magda Gasull 1,3,4, Miquel Porta 1,3,4,*; PANKRAS II Study Group**
PMCID: PMC6786909  NIHMSID: NIHMS1028819  PMID: 31066938

Abstract

Trace elements are a possible risk factor for pancreatic ductal adenocarcinoma (PDAC). However, their role in the occurrence and persistence of KRAS mutations remains unstudied. There appear to be no studies analyzing biomarkers of trace elements and KRAS mutations in any human cancer. We aimed to determine whether patients with KRAS mutated and non-mutated tumours exhibit differences in concentrations of trace elements. Incident cases of PDAC were prospectively identified in five hospitals in Spain. KRAS mutational status was determined through polymerase chain reaction from tumour tissue. Concentrations of 12 trace elements were determined in toenail samples by inductively coupled plasma mass spectrometry. Concentrations of trace elements were compared in 78 PDAC cases and 416 hospital-based controls (case-control analyses), and between 17 KRAS wild-type tumours and 61 KRAS mutated tumours (case-case analyses). Higher levels of iron, arsenic, and vanadium were associated with a statistically nonsignificant increased risk of a KRAS wild-type PDAC (OR for higher tertile of arsenic = 3.37, 95% CI: 0.98–11.57). Lower levels of nickel and manganese were associated with a statistically significant higher risk of a KRAS mutated PDAC (OR for manganese = 0.34, 95% CI: 0.14–0.80). Higher levels of selenium appeared protective for both mutated and KRAS wild-type PDAC. Higher levels of cadmium and lead were clear risk factors for both KRAS mutated and wild-type cases. This is the first study analyzing biomarkers of trace elements and KRAS mutations in any human cancer. Concentrations of trace elements differed markedly between PDAC cases with and without mutations in codon 12 of the KRAS oncogene, thus suggesting a role for trace elements in pancreatic and perhaps other cancers with such mutations.

Keywords: Pancreatic ductal adenocarcinoma, Pancreatic neoplasm, Trace elements, KRAS oncogene, Etiology

INTRODUCTION

In spite of decades of research, the etiology of pancreatic ductal adenocarcinoma (PDAC) remains poorly understood [Kamisawa et al. 2016]. Research on environmental and other modifiable causes could lead to more effective primary prevention. Lifestyle risk factors include tobacco smoking, heavy alcohol intake and obesity [Hart et al. 2008]. Among environmental factors, PDAC has been linked with exposure to aromatic amines and to several trace elements such as arsenic, cadmium and lead [Amaral et al. 2012; Antwi et al. 2015; Chen et al. 2015; Hart et al. 2008; Luckett et al. 2012; Porta et al. 1999].

In a previous study, we showed that higher toenail concentrations of cadmium, lead and arsenic significantly increased the risk of PDAC. In contrast, higher levels of selenium and nickel were protective [Amaral et al. 2012]. Several studies have identified cadmium exposure as a risk factor for PDAC [Amaral et al. 2012; Chen et al. 2015; Luckett et al. 2012; Kriegel et al. 2006]. Higher selenium levels were also protective against PDAC and were associated with longer survival [Lener et al. 2016].

Most trace elements are considered weak mutagens, and non-genotoxic mechanisms are their predominant carcinogenic mode of action. In brief, trace elements induce oxidative stress, defective DNA repair, genomic instability, post-translational histone modifications, and altered methylation of tumour-suppressor genes and oncogenes [Chervona et al. 2012; Henkler and Luch 2011; Henkler et al. 2010; Hernández et al. 2009; Luch 2005; Stein 2012].

Mutations in the KRAS oncogene play an important role in several neoplasms [Deramaudt and Rustgi, 2005; Eser et al. 2014; Waddell et al. 2015]. They are an early event in the carcinogenic process; this fact provides a rationale to seek reasons for their occurrence and persistence. In PDAC, codon 12 mutations are identified in 70 to 90% of tumours [Deramaudt and Rustgi, 2005; Porta et al. 1999; Waddell et al. 2015]. Although oncogenic KRAS signalling may be crucial to the progression and maintenance of the most frequent form of PDAC, some tumours do not harbour KRAS mutations [Bittoni et al. 2015; Collisson et al. 2011, 2012; Deramaudt and Rustgi, 2005; Porta et al. 1999; Schlitter et al. 2017]. Wild-type KRAS PDAC has distinct morphological variants and gene expression patterns [Bittoni et al. 2015; Schlitter et al. 2017]. Different causal processes, perhaps including different gene-environment interactions, may influence the development of KRAS wild-type and mutated PDAC [Bittoni et al. 2015; Collisson et al. 2011, 2012; Crous-Bou 2009; Crous-Bou et al. 2009; Porta et al. 1999, 2003; Schlitter et al. 2017]. Non-genotoxic mechanisms like epigenetic events and cytotoxicity may play a greater role in the genesis and progression of KRAS wild-type tumours [O’brien et al. 2013]. There appear to be no studies analyzing biomarkers of trace elements and KRAS mutations in any human cancer.

Thus, our hypothesis was that higher concentrations of non-genotoxic carcinogens, such as some trace elements, would be found in patients with KRAS wild-type PDAC than in patients with KRAS mutated PDAC. Consequently, our main objective was to compare concentrations of trace elements in PDAC cases with KRAS wild-type and mutated tumours. To assess the results of such comparison, we also analyzed concentrations of trace elements in a group of controls free from pancreatic cancer.

MATERIALS AND METHODS

Study Population

We analyzed 78 incident PDAC cases from the PANKRAS II Study and 416 hospital-based controls from the EPICURO Study [Amaral et al. 2012; Porta et al. 1999; Garcia-Closas et al. 2005]. The two studies had overlapping geographical recruitment areas and were performed close in time. Controls, recruited during 1998 to 2001, were patients with diagnoses unrelated to the exposures of interest [Amaral et al. 2012; Garcia-Closas et al. 2005]. In the PANKRAS II study [Alguacil et al. 2002, 2003; Crous-Bou 2009; Crous-Bou et al. 2009; Parker et al. 2011b; Porta et al. 1999, 2000, 2005, 2009b; Soler et al. 1999], subjects were recruited between 1992 and 1995 at five general hospitals in Eastern Spain, where 185 incident cases of PDAC were prospectively identified. All their diagnoses were reviewed by a panel of experts and by the study reference pathologists, blinded to the original diagnoses and to molecular results [Porta et al. 2000; Soler et al. 1999].

Information on both mutations in codon 12 of the KRAS gene and concentrations of trace elements in toenails was available for 78 cases, of whom 61 (78.2%) had KRAS mutations and 17 did not. The present report is thus based on this subset of patients with known KRAS mutational status and levels of trace elements. There were no significant differences between this subgroup and the remaining PDAC cases with respect to sex, education, tumour stage, consumption of coffee, tobacco smoking, or alcohol intake. Nonetheless, the 78 patients were slightly younger, and had a slightly higher frecuency of the constitutional syndrome at presentation than the patients excluded because they lacked information on either KRAS mutations or on trace elements (see Supporting Information Table I) [Alguacil et al. 2002, 2003; Porta et al. 2009b]. Informed consent from all subjects and ethical approval from local institutional review boards were obtained.

Clinicopathological Information and Personal Interviews

More than 97% of PDAC cases were interviewed face-to-face by trained monitors during hospital stay, close to the time of diagnosis. Interviews included questions about past clinical history, signs and symptoms of pancreatic cancer, occupation, diet and coffee, alcohol and tobacco consumption [Crous-Bou 2009; Crous-Bou et al. 2009; Parker et al. 2011b; Porta et al. 1999, 2005; Soler et al. 1999]. A structured form was used to collect clinicopathological information from medical records, including details on diagnostic procedures, semiology, tumour stage, laboratory results and follow-up [Crous-Bou et al. 2009; Parker et al. 2011b; Porta et al. 2000, 2005; Soler et al. 1999].

There were no significant differences in sociodemographic and clinicopathological information between KRAS mutated and wild-type cases (see Supplementary Table II). Cases with KRAS wild-type tumors differed in some characteristics from cases with KRAS mutant tumors. Specifically, wild-type KRAS cases were less often regular coffee drinkers, had a higher percentage of tumours located in the tail of the organ, and slightly more often diabetes mellitus, consistent with findings from previous analyses in the larger set of cases with known KRAS status [Crous-Bou 2009; Porta et al. 2009b]. There were no significant differences between KRAS mutated and wild-type cases regarding age, sex, education, occupation, consumption of alcohol and tobacco, energy intake, tumour stage, time from first symptom to diagnosis and toenail clipping, clinical presentation or hospital of admission (Supplementary Table II).

Detection of KRAS Mutations

Cytohistological samples from patient tumors were obtained during hospital stay. Details of tissue specimens and laboratory protocols have been described in detail elsewhere [Alguacil et al. 2002, 2003; Berrozpe et al. 1994; Crous-Bou 2009; Malats et al. 1995, 1997; Porta et al. 1999a, 1999b, 2009b]. Briefly, mutations in codon 12 of the KRAS oncogene were studied using DNA extracted from paraffin-embedded tumour tissue. Tumor DNA was extracted and analyzed immediately after the end of patient recruitment (i.e, from a few weeks to about three years after tumor procurement). Amplifications were done in two steps by nested polymerase chain reaction; in the second amplification reaction, an artificial BstNI restriction endonuclease site was created to discriminate between wild-type and mutated KRAS codon 12 sequences. The 103 bp product of this amplification reaction was digested overnight. Wild-type sequences were cleaved, resulting in two fragments of 82 and 21 bp, whereas codon 12 mutated sequences were not. Products were analyzed by acrylamide gel electrophoresis and ethidium bromide staining. This technique was able to detect 1 heterozygously mutated cell per 50 wild-type cells (i.e., 1 mutant allele per 50 wild-type alleles). To characterize the nucleotide substitution in codon 12, all mutated samples were further analyzed using a similar restriction fragment length polymorphism-approach, as described elsewhere (Berrozpe et al. 1994; Malats et al. 1995, 1997). Interpretation of digestion products’ electrophoresis was performed independently by two investigators to confirm the results. When discordant results were obtained, the analysis was repeated and results evaluated again. This strategy has been shown to yield an agreement of >95% for all enzyme digestions (Malats et al. 1995, 1997).

Trace Element Assessment

Toenail samples were also obtained during hospital stay [Amaral et al. 2012]. Toenails are not altered with long-term storage, and they are a valid measure of cumulative exposure to trace elements 6–18 months prior to their clipping [He 2011; Laohaudomchok et al. 2011; Slotnick and Nriagu 2006; Slotnick et al. 2007; Wickre et al. 2004]. The rationale for their use in the present study is that the ranking of toenail concentrations in a group of individuals (e.g., in KRAS mutated and non-mutated cases, and controls) at one point in time will be highly similar to the ranking in the more distant past.

Toenails were stored at room temperature until analysis. In 2009 trace elements were quantified at the Trace Element Analysis Core (Dartmouth College, NH, USA) using inductively coupled plasma-mass spectrometry. Toenails were acid digested with Optima nitric acid (Fisher Scientific, St. Louis, MO) at 105°C followed by addition of hydrogen peroxide and further heating the dilution with deionized water. All sample preparation steps were recorded gravimetrically. As a quality control, each batch of analyses included six standard reference material samples with known trace element concentrations and six analytic blanks, along with the study samples. The case-control status of study participants was not disclosed to the testing laboratory [Amaral et al. 2012].

Statistical Analysis

The 78 PDAC cases and the 416 controls were divided into tertiles based on the distribution of trace element concentrations among controls. For these comparisons, we used multivariate-adjusted odds ratios (aOR) and their corresponding 95% confidence intervals (CI), calculated by unconditional logistic regression. To further assess the relation of trace elements with KRAS mutations we performed case-case analyses considering potential confounders [Amaral et al. 2012; Crous-Bou 2009; Porta et al. 1999a, 1999b].

Univariate statistics were computed as customary [Armitage et al. 2001]. To estimate the magnitude of the associations between trace element concentrations and KRAS mutations, we also used aORs, and their corresponding 95% CIs, calculated by unconditional logistic regression. Categorical ordinal variables were analyzed for a linear dose–response relation through the multivariate analogue of Mantel-Haenszel’s extension test; when a linear trend was not apparent, Wald’s test was used. Age, sex, and tobacco consumption were assessed in all models as potential confounders. Other possible confounding variables were also retained in the models when they materially altered the estimates; such confounders included coffee and alcohol intake, geographical region, and tumour stage. Final models were chosen coherently with the nature of the variables and the study objectives. The level of statistical significance was set at 0.05 and all tests were two tailed. Statistical analyses were performed using SPSS version 18 (SPSS, Armonk, NY, USA, 2009) and R version 3.2.1 (R Core Team, Vienna, Austria, 2015).

RESULTS

Concentrations of Trace Elements

KRAS mutated and wild-type PDAC cases had significantly different concentrations of several trace elements. Cases with a KRAS wild-type had overall higher median levels of trace elements while KRAS mutated cases showed decreased concentrations for most elements (Fig. 1 and Table I).

Fig. 1.

Fig. 1.

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Unadjusted comparison of trace element concentrations in hospital controls, KRAS wild-type cases and KRAS mutated cases. Letters A to E classify trace elements into 5 patterns according to their relative levels across groups. Bar graph shows median value, brackets over bars indicate groups significantly different (Mann Whitney’s U test). *P<0.05, **P<0.01, ***P<0.001.

Table I.

Trace element concentrations (μg/g) in PDAC cases by KRAS status: wild-type vs. mutated.


 KRAS status
Total Wild-type Mutated
Trace elements (μg/g) N (%) N (%) N (%) P
Total 78 (100) 17 (21.8) 61 (78.2)
Selenium
 Mean ± standard deviation 0.51 ± 0.12 0.56 ± 0.09 0.49 ± 0.12 0.039a
 Median 0.51 0.55 0.49 0.040b
 Minimum - Maximum 0 – 0.77 0.42 – 0.77 0 – 0.73
Manganese
 Mean ± standard deviation 0.47 ± 0.72 0.80 ± 1.01  0.37 ± 0.59 0.109a
 Median 0.22 0.26 0.22 0.104b
 Minimum - Maximum 0.05 – 3.93 0.11 – 3.22 0.05 – 3.93
Aluminium
 Mean ± standard deviation 13.28 ± 16.40 22.37 ± 24.72  10.74 ± 12.34 0.077a
 Median 7.55 9.12 7.38 0.026b
 Minimum - Maximum 1.35 – 92.33 4.46 – 75.04 1.35 – 92.33
Zinc
 Mean ± standard deviation 119.8 ± 50.05 142.4 ± 56.77  113.5 ± 46.58 0.067a
 Median 107.2 118.7 105.4 0.010b
 Minimum - Maximum 35.54 – 420.8 96.08 – 285.4 35.54 – 420.8
Iron
 Mean ± standard deviation 21.28 ± 25.73 36.41 ± 41.75 17.06 ± 17.37 0.079a
 Median 12.68 16.11 12.03 0.040b
 Minimum - Maximum 6.32 – 128.6 8.66 – 128.6 6.32 – 127.6
Vanadium
 Mean ± standard deviation 0.04 ± 0.09 0.09 ± 0.19  0.03 ± 0.03 0.224a
 Median 0.02 0.03 0.02 0.026b
 Minimum - Maximum 0.01 – 0.79 0.01 – 0.79 0.01 – 0.19
Arsenic
 Mean ± standard deviation 0.11 ± 0.09 0.12 ± 0.08 0.10 ± 0.09 0.500a
 Median 0.09 0.10 0.08 0.355b
 Minimum - Maximum 0 – 0.55 0.03 – 0.31 0 – 0.55
Copper
 Mean ± standard deviation 3.92 ± 1.84 4.78 ± 2.80 3.69 ± 1.40 0.137a
 Median 3.58 3.85 3.44 0.210b
 Minimum - Maximum 1.24 – 13.59 2.52 – 13.59 1.24 – 10.59
Chromium
 Mean ± standard deviation 0.76 ± 1.09 1.21 ± 2.00 0.63 ± 0.62 0.258a
 Median 0.43 0.45 0.43 0.342b
 Minimum - Maximum 0.09 – 8.49 0.09 – 8.49 0.09 – 3.65
Nickel
 Mean ± standard deviation 0.78 ± 3.79 2.58 ± 8.02 0.27 ± 0.26 0.253a
 Median 0.22 0.51 0.20 0.009b
 Minimum - Maximum 0.01 – 33.58 0.06 – 33.58 0.01–1.39
Cadmium
 Mean ± standard deviation 0.06 ± 0.09 0.10 ± 0.15 0.04 ± 0.06 0.156a
 Median 0.03 0.04 0.03 0.086b
 Minimum - Maximum 0 – 0.62 0 – 0.62 0 – 0.33
Lead
 Mean ± standard deviation 3.16 ± 17.65 1.66 ± 1.35 3.58 ± 19.97 0.694a
 Median 0.86 1.13 0.79 0.030b
 Minimum - Maximum 0.17 – 156.8 0.31 – 5.48 0.17 – 156.8
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a

Student’s t-test (two-tailed).

b

Mann-Whitney’s U test (two-tailed).

Five patterns of crude (unadjusted) concentrations of trace elements were identified (labels A to E in Fig. 1). Selenium, manganese and aluminium levels were highest in controls, intermediate in PDAC cases with KRAS wild-type, and lowest in PDAC cases with mutated KRAS. Median concentrations of zinc, iron and vanadium were statistically significantly higher in KRAS wild-type cases than in KRAS mutated cases. Although levels of arsenic, copper, and chromium were not statistically different among groups, KRAS wild-type cases exhibited higher median levels than KRAS mutated cases and controls. Nickel showed statistically significantly lower levels in KRAS mutated cases than in KRAS wild-type cases and controls. Finally, both KRAS mutated and wild-type cases had statistically significantly higher levels of cadmium and lead in comparison to controls. Lead was also statistically significantly higher in KRAS wild-type cases in comparison to their KRAS mutated counterparts (Fig. 1E).

The highest correlations between pairs of elements among cases were: vanadium and manganese (rho = 0.680), vanadium and iron (rho = 0.667), manganese and iron (rho = 0.642), aluminum and iron (rho = 0.618), and aluminum and vanadium (rho = 0.568) (Supplementary Fig. 1).

Case–Control Comparison

There were marked differences in the aORs for KRAS mutated and wild-type PDAC cases vs. controls (middle and right columns of Fig. 2). As expected, because 78% of cases had KRAS mutated tumours, the aORs for all cases vs. controls were very similar to those for KRAS mutated cases vs. controls.

Fig. 2.

Fig. 2.

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Associations between pancreatic cancer risk (mutated and wild-type KRAS) and concentrations of trace elements. aOR (highest tertile vs. lowest) and p-value for linear trend. *P<0.05, **P<0.01, ***P<0.001. Bold typeface indicates P<0.05, red shading an aOR>1 and green shading an aOR<1. All estimates are adjusted for age, sex and smoking status (ever/never).

Higher concentrations of selenium were associated with an statistically significant lower risk of developing both mutated and wild-type KRAS PDAC (aOR=0.07, 95% CI 0.02–0.21 and aOR=0.23, 95% CI 0.06–0.94, respectively). Higher levels of nickel were inversely associated with a KRAS mutated PDAC (aOR= 0.13, 95% CI 0.05–0.34). Aluminium, manganese and zinc displayed similar protection against KRAS mutated neoplasms (Fig. 2 and Supplementary Table III).

In contrast, higher levels of iron and vanadium were associated with a remarkable, though statistically nonsignificant increased probability of a wild-type KRAS tumour (aOR for the highest tertile of iron = 5.30, 95% CI 0.98–28.78). Higher concentrations of arsenic were also associated with a higher chance of developing a KRAS wild-type tumour (aOR = 3.37, 95% CI 0.98–11.57), while the risk of developing a KRAS mutated tumour was only slightly increased (Fig. 2 and Supplementary Table III).

Cadmium and lead were significant risk factors for both KRAS mutated and wild-type cases. PDAC risk due to cadmium exposure was greater for women than men (in women, aOR for the highest tertile = 6.77, 95% CI 1.96–23.36, p for trend = 0.002, and in men, aOR = 2.67, 95% CI 1.19–5.98, p for trend = 0.008). Cadmium was similarly associated with KRAS mutated and wild-type PDAC: aOR for the highest tertile = 3.39 (95% CI 1.59–7.2), and 4.47 (95% CI 1.20–16.6), respectively. Higher levels of lead were associated with a greater risk of developing a KRAS mutated neoplasm, and with an even greater risk of developing a KRAS wild-type neoplasm (aOR = 21.6, 95% CI 2.64–176) (Fig. 2 and Supplementary Table III).

When concentrations of selenium were included in the models, the association between lead and KRAS mutated PDAC increased (aOR = 7.84, 95% CI 2.72–22.59). It also increased the association between lead and KRAS wild-type PDAC (aOR = 39.4, 95% CI 3.80–409). The associations with cadmium remained statistically significant and slightly weaker. Also, the association between arsenic and KRAS wild-type PDAC became stronger and statistically significant when selenium concentrations were taken into account (aOR for the highest tertile = 4.21, 95% CI 1.14–15.58).

Case–Case Comparison

In multivariate logistic regression analyses between PDAC cases, higher concentrations of manganese were associated with a significant higher odds of having a tumour without KRAS mutations (Table II). Manganese was found in lower levels in cases with KRAS mutations than in cases without KRAS mutations. Although not statistically significant in all models, higher concentrations of aluminium were also associated with a greater probability of having a wild-type tumour (see Table II and Supplementary Table IV).

Table II.

Effect of selected trace elements (μg/g) in tertiles on the probability of a KRAS wild-type (vs. mutated) pancreatic ductal adenocarcinoma.

Trace elements Unadjusted model Adjusted models
(μg/g) OR (95% CI) Pa aORb (95% CI) Pa aORc (95% CI) Pa
Manganese
 ≤0.17 1.00 Ref. 0.048 1.00 Ref. 0.036 1.00 Ref. 0.014
 0.18–0.44 0.96 (0.25–3.72) 0.90 (0.16–5.17) 1.19 (0.18–8.01)
 >0.44 4.55 (1.13–18.35) 8.57 (1.36–53.87) 18.07 (2.09–156.5)
Aluminium
 ≤7.10 1.00 Ref. 0.021 1.00 Ref. 0.065 1.00 Ref. 0.051
 7.11–16.75 3.23 (0.79–13.25) 3.10 (0.64–15.18) 2.82 (0.57–14.11)
 >16.75 6.67 (1.28–34.84) 5.10 (0.83–31.24) 6.40 (0.96–42.77)
Zinc
 ≤98.23 1.00 Ref. 0.015 1.00 Ref. 0.019 1.00 Ref. 0.009
 98.24–117.62 2.64 (0.48–14.45) 5.90 (0.58–59.99) 6.81 (0.60–77.69)
 >117.62 7.07 (1.33–37.65) 12.09 (1.27–115.1) 22.28 (1.85–268.8)
Iron
 ≤9.92 1.00 Ref. 0.087 1.00 Ref. 0.180 1.00 Ref. 0.075
 9.93–21.20 2.61 (0.51–13.45) 1.28 (0.20–8.34) 1.17 (0.16–8.79)
 >21.20 4.50 (0.77–26.13) 3.46 (0.49–24.67) 6.77 (0.75–61.04)
Nickel
 ≤0.23 1.00 0.040 1.00 0.027 1.00 0.016
 0.24–0.64 1.80 (0.46–6.98) 2.76 (0.59–12.93) 2.93 (0.61–14.11)
 >0.64 10.1 (2.29–44.31) 8.88 (1.27–62.21) 15.89 (1.70–148.4)
Lead
 ≤0.32 1.00 Ref. 0.038 1.00 Ref. 0.059 1.00 Ref. 0.047
 0.33–0.66 0.32 (0.02–5.78) 0.21 (0.01–5.61) 0.38 (0.01–12.08)
 >0.66 3.28 (0.37–29.12) 2.98 (0.25–35.2) 4.31 (0.31–59.59)
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OR: odds ratio; CI: confidence interval; aOR: adjusted odds ratio; Ref.: Reference.

a

p-value derived from multivariate analogue of Mantel’s extension test for linear trend.

b

Odds ratio adjusted for age, sex, region, tobacco smoking (ever/never), alcohol consumption

and coffee intake (cups per week).

c

Odds ratio further adjusted for tumour stage.

Higher concentrations of zinc, nickel and lead were more strongly associated with KRAS wild-type tumours than with KRAS mutated tumours in all models (Table II). Adjusting for potential confounders as age, sex and smoking did not significantly alter the estimates, except for manganese and zinc (Table II). After adjusting for tumour stage, the estimates increased for most trace elements (Table II and Supplementary Table IV). Higher concentrations of cadmium and selenium were not associated with KRAS mutational status (Supplementary Table IV).

DISCUSSION

Concentrations of trace elements differed markedly between PDAC cases with and without mutations in codon 12 of the KRAS oncogene. Furthermore, the inclusion of controls unveiled five possible patterns of association (Fig. 1A–E) in what is the first study using biomarkers of trace elements to assess potential relationships between such compounds and KRAS mutations in any human cancer.

Concentrations of selenium, manganese and aluminium were highest in controls, intermediate in wild-type KRAS PDAC patients and lowest in patients with KRAS mutations. In case-control comparisons selenium appeared as an important protective factor for PDAC regardless of KRAS mutational status. These results agree with other studies [Amaral et al. 2012; Lener et al. 2016]. The findings are also supported by selenium’s role in antagonizing the oxidative effects of arsenic, cadmium, and lead, and boosting the gatekeeping activity of p53 [Fowler et al. 2004; Jackson and Combs 2008; Schrauzer et al. 2000; Smith et al. 2004]. After adjusting by selenium in our models, we found that arsenic and lead were more strongly associated with a KRAS wild-type tumour, and in the case of lead, also with a KRAS mutated PDAC. Evidence on associations between KRAS mutations, downstream cell signaling, and oxidative stress should be considered when assessing these and related findings discussed below [Parsons et al 2013; Boldogh et al. 2012]. However, it is beyond the scope of our observational study in humans to address in detail such mechanistic scenarios.

In case-control comparisons, lower concentrations of manganese and aluminium were associated with a mutated KRAS PDAC. Manganese deficiency has been linked to lower activities of manganese-dependent superoxide dismutase (MnSOD) [Finley and Davis 1999; Li et al. 2016]. This enzyme reduces oxidative stress by catalysing the conversion of superoxide to hydrogen peroxide and water [Finley and Davis 1999]. Therefore, a possible explanation is that lower manganese levels may account for an increase in oxidative stress, favouring the occurrence and persistence of KRAS mutations [Li et al. 2016]. The association between lower concentrations of aluminium and mutated KRAS PDAC may be explained by the high correlation between aluminum and manganese (Supplementary Fig. 1).

Zinc, iron and vanadium were at significantly higher concentrations amongst patients without KRAS mutations, and adjusted case-case analyses revealed a strong association between higher levels of zinc and having a KRAS wild-type tumour. Zinc is an essential trace element with antioxidant and genome-stabilizing functions [Alam and Kelleher 2012; Eide 2011]. This suggests that higher levels of zinc may protect against the acquisition or persistence of KRAS codon 12 mutations in PDAC. Higher concentrations of iron only entailed an increased risk of developing a wild-type KRAS PDAC. Although barely non-statistically significant, this result may deserve further studies because iron has not been previously identified as a potential risk factor for PDAC. Iron is a known carcinogen: it promotes mitosis, interferes with MnSOD functionalism, activates xanthine oxidase causing oxidative damage, inhibits apoptosis, and increases telomerase activity [Toyokuni 2009; Yamamoto et al. 2016]. As with iron, higher concentrations of vanadium were associated with a higher probability of having a PDAC without KRAS mutations. Vanadium induces genotoxic lesions, inhibits apoptosis and favours cell transformation [Assem and Levy 2009; Zwolak 2014].

Arsenic, chromium and copper were all found in slightly higher concentrations in PDAC cases without KRAS mutations. Arsenic had been previously identified as a risk factor for PDAC [Amaral et al. 2012]; in our study, this increased risk was only significant for patients with KRAS wild-type tumours. Consequently, arsenic might play a greater carcinogenic role in patients whose tumours do not harbour KRAS mutations. Arsenic is a potent inductor of oxidative stress and DNA damage, and is also known to interact with DNA repair enzymes [Fowler et al. 2004; Henkler et al. 2010; Muenyi et al. 2015; Reichard and Puga 2010].

Nickel concentrations were highest in KRAS wild-type cases, intermediate in controls and lowest in KRAS mutated cases. In case-case analyses, patients with the highest concentrations of nickel were 15 times more likely to have a tumour without KRAS mutations than with such mutations. Nickel’s carcinogenic effects are probably caused by epigenetic alterations and induction of oxidative stress [Ellen et al. 2009; Henkler et al. 2010]. Intracellular nickel is known to induce hypoxia inducible factor 1 (HIF-1) promoting cell proliferation and up-regulation of p53 leading to growth arrest. This may lead to a selective pressure of cells harbouring mutations in tumour supressor genes and not necessarily in oncogenes such as KRAS [Henkler et al. 2010].

Cadmium and lead were particularly high in KRAS wild-type cases, lower in KRAS mutated cases and much lower in controls. This pattern is in line with studies that identified cadmium and lead as important risk factors for developing PDAC [Amaral et al. 2012; Chen et al. 2015; Kriegel et al. 2006; Luckett et al. 2012]. For cadmium, both KRAS mutational situations exhibited similar adjusted odds ratios in case-control comparisons. Therefore, cadmium-induced PDAC may be independent of KRAS codon 12 mutations. Cadmium’s mode of action involves stimulating cell proliferation, transdifferentiation of pancreatic cells, acting as a mitogen, and inhibiting DNA repair and apoptosis [Hartwig 2010; Luevano and Damodaran 2014].

Similarly, higher levels of lead increased the risk of both of KRAS mutated and wild-type PDAC, thus suggesting that lead may be a risk factor for PDAC independent of mutations in codon 12 of KRAS. Lead may also act through the induction of chromosome aberrations, micronuclei and sister chromatid exchanges [IARC 2006; Wu et al. 2012].

A rationale exists for overall higher levels of toxic trace elements in patients with KRAS wild-type PDAC, and for lower levels of protective trace elements in KRAS mutated tumours. In a previous study we showed that wild-type KRAS PDAC was more likely to arise in subjects with chronic pancreatitis and multiple medical conditions [Crous-Bou et al. 2009]. Such findings suggested that inflammatory and non-genotoxic mechanisms, the main mode of action of most trace elements, may be the predominant carcinogenic pathways for wild-type KRAS PDAC [O’brien et al. 2013]. In contrast, KRAS mutated PDAC has been associated with higher levels of organochlorine compounds. Perhaps due to the selective advantage provided by KRAS mutations, tumour cells may be less influenced by other carcinogenic agents such as trace elements [Collisson et al. 2012; Deramaudt and Rustgi 2005; Eser et al. 2014; Porta et al. 1999a; Schlitter et al. 2017]. Decreased levels of selenium and manganese in patients with KRAS mutated tumours may be explained by their established antioxidant and gatekeeping properties, therefore suggesting a potentially important role in preventing the acquisition or persistence of KRAS mutations in PDAC [Finley and Davis 1999; Jackson and Combs 2008; Li et al. 2016; Schrauzer et al. 2000; Smith et al. 2004]. The previous considerations notwithstanding, it is also important to consider how KRAS mutations participate in and are themselves impacted during tumor progression. KRAS mutation is an early driver of a large percentages of PDAC [Waters and Der 2018], but KRAS mutant cells may be selected against during tumor progression [Parsons and Myers 2013].

The strengths of our study include the recruitment of incident PDAC cases, excellent information on potential confounders, and the biological measurement of trace element in toenails. The latter feature is particularly relevant, as most studies have determined trace elements in serum or in pancreatic juice at the time of diagnosis [Carrigan et al. 2007; Farzin et al. 2013; Kriegel et al. 2006; Laohaudomchok et al.2011; Lener et al. 2016]. No such studies assessed KRAS mutations.

Lipid mobilization and the other metabolic changes that commonly characterize cancer progression are unlikely to have influenced toenail levels of the trace elements. Unlike blood and urine, hair and nail samples reflect the concentration of elements in the organism over several months, and are thus useful for the evaluation of chronic exposure [Golasik et al. 2015; Goyer and Clarkson 2001; He 2011; Hopps 1977; Slotnick and Nriagu 2006]. Trace elements in nails are incorporated during their formation (12 – 18 months) from blood, lymph vessels, body tissues and epidermis; thus, they reflect exposures that have occurred in such months [Goyer and Clarkson 2001; Hopps 1977; Slotnick and Nriagu 2006]. However, the rationale for their use in studies on disease etiology is that the ranking of toenail concentrations in a group of individuals at one point in time (e.g., at diagnosis) will be highly similar to the ranking in the more distant past (e.g., during the relevant phases of tumor biological onset) [Golasik et al. 2015; Goyer and Clarkson 2001; He 2011; Hopps 1977; Slotnick and Nriagu 2006].

Although controls were recruited around 3 years after the identification of PDAC cases, the control group was matched by geographical region and had a similar age distribution. Spain banned leaded fuels in 2002, shortly after the recruitment of controls, but exposure had been decreasing for some years [Amaral et al. 2012; IARC 2006].

Another strength of the study is the assessment of KRAS mutations in tumour tissue, which is known to yield better sensitivity than detection in serum [Brychta et al. 2016; Parker et al. 2001a, 2011b; Takai et al. 2015]. However, polymerase chain reaction and restriction fragment length polymorphism analysis yield a lower sensitivity to detect KRAS mutations than more modern techniques as droplet digital (dd) PCR, high resolution melting analysis or next-generation sequencing [Schlitter et al. 2017]. Thus, many or perhaps all samples we characterized as wild-type probably had undetected KRAS mutations. Therefore, tumors identified as KRAS wild-type and KRAS mutant likely had levels of KRAS mutations less than and greater than 1 in 50 alleles, respectively.

Low numbers of cases prevented us from analyzing the different KRAS codon 12 mutations (i.e., their spectrum), nor did we assess KRAS codon 13 mutations (which are uncommon in PDAC). Different KRAS mutations have different downstream signaling effects, do not necessarily confer the same phenotype [Céspedes et al. 2006], and, more importantly in our study, their occurrence and persistence may be influenced by different environmental compounds [Porta et al. 2003, 2009a; Shields et al. 2000].

Our sample size was undoubtedly small, and estimates were often statistically imprecise or statistically nonsignificant. It is clear that results must be replicated or refuted. However, as mentioned, this is the largest study analyzing biomarkers of trace elements and KRAS mutations in any human cancer. Also, we present detailed information on the few differences between cases included and cases excluded because they lacked information on either KRAS mutations or on trace elements. In spite of the low statistical power to detect weak associations, our data did have enough power to detect associations of substantial magnitude. Multiple comparisons were made, with the ensuing risk of false positives. Results definitely warrant confirmation in longitudinal studies with larger sample sizes. If the roles of trace elements in the etiology of pancreatic ductal adenocarcinoma were confirmed, individual measures (e.g., through nutrition), and public policies to reduce population exposure would gain support. And new knowledge would be available on the role of trace elements in the prevention and development of pancreatic and other tumors with KRAS mutations.

Supplementary Material

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ACKNOWLEDGEMENT

The authors gratefully acknowledge technical and scientific assistance provided by Yolanda Rovira and María Gómez. The work was supported in part by research grants from Instituto de Salud Carlos III, Ministry of Health, Government of Spain (FIS PI13/00020, FIS PI17/00088, and CIBER de Epidemiología y Salud Pública - CIBERESP); the Hospital del Mar Medical Research Institute (IMIM), Barcelona; Fundació La Marató de TV3 (20132910); the Government of Catalonia (2014 SGR 1012, 2017 SGR 439); the Association for International Cancer Research (AICR09–0780); and the Intramural Research Program of the Division of Cancer Epidemiology and Genetics, National Cancer Institute, USA. The authors have no conflicts of interest in connection with the paper, and declare no competing financial interests.

Abbreviations:

aOR

adjusted odds ratio

CI

confidence interval

MnSOD

manganese-dependent superoxide dismutase

OR

odds ratio

PDAC

pancreatic ductal adenocarcinoma

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