TP53 Arg72Pro Polymorphism and Pancreatic Cancer Susceptibility: Assessing rs1042522 as a Risk Factor for Pancreatic Cancer

1. Background

Pancreatic cancer (PaC) is the 4th cancer-related cause of death in Western countries and is estimated to become the 2nd by 2030 [1]. It has a high mortality rate since only a small percentage of patients are diagnosed in a resectable, hence curable, stage. Approximately 80% of patients are not amenable to resection at diagnosis due to metastatic disease or local infiltration, and another 41.4% of patients candidate to surgery have unresectable tumors found during exploratory laparoscopy/tomy [2]. The economic burden of a hypothetic pancreatic screening restrains from offering a second level imaging to populations with increased odds ratio, from 1.7 to 2.2, for pancreatic cancer. This includes those affected by obesity, pancreatitis and diabetes [3]. Pancreatic cancer screening in asymptomatic patients is impractical. To detect a case, one should screen over 17000 people and to find a resectable, hence curable, patient, over 35000 people should be screened [4]. Finding new genomic susceptibilities to PaC in these high risk populations might narrow the field for early diagnosis.

This study is part of a larger one focusing on the genomic and somatic landscape of the four major driver genes involved in pancreatic cancerogenesis: KRAS, TP53, SMAD4, CDKN2A.

In particular, we focused on the analysis of TP53 polymorphism in a PaC cohort compared to a reference healthy population.

2. Methods

The Institutional Review Board of the University of Sapienza approved the protocol (Prot. 243 SA_2018, 12/12/2018), where every participating center had to subscribe to the IRB. As per the Helsinki Declaration, the protocol was registered at clinicaltrials.gov with the identification number NCT03524677. All consecutive naive patients with non-metastatic PaC who are applicable for upfront pancreatic resection have been enrolled on a multi-center basis from 2017 to 2020. The enrollment took place in 3 institutions: University Hospital Sant’Andrea (Rome), the promoter, University Hospital A. Gemelli (Rome) and Community Hospital G. Salvini (Garbagnate Milanese, MI). After a signed informed consensus, blood samples in EDTA were preoperatively collected and centrifuged twice within 1 hour at 2300 rpm for 10 minutes. Plasma that was obtained was preserved and stored at -80C°. A protected anonymous multi parametric database was filled with relevant information about the patients and disease. Circulating free DNA (cfDNA) was extracted from 1 to 3mL of plasma samples using a Norgen RNA/DNA purification kit as indicated by the manufacturer protocol procedure and sequenced with a Myriapod^® NGS 56G Onco panel (Diatech Pharmacogenetics) on the MiniSeqTM Illumina platform for the analysis of hot-spot mutations of 56 genes. The panel allowed us to obtain 263 amplicons, with lengths from 92 to 184 bases, covering the hot-spots and surrounding regions.

A comparison with a reference population was then carried out with data from 1000GenomeProject [5] on healthy subjects from central Italy (Tuscany).

The statistical analysis has been carried out with the online Odds Ratio Calculator [MedCalc Software Ltd. Odds ratio calculator. https://www.medcalc.org/calc/odds_ratio.php (Version 20.115; accessed September 21, 2022)].

This study has been reported according to the STrengthening the REporting of Genetic Association studies (STREGA) which is an extension of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement [6].

3. Results

3.1. TP53 Polymorphism

Patients characteristics are listed in Table 1. Next generation sequencing revealed the rs1042522 variant of gene TP53 in 30 out of 35 patients. In 4 patients it yielded a low coverage (<500 reads) with a variant frequency of >99%, therefore making them included in the analysis. In all 30 patients with rs1042522, a cytosine (C) was replaced by a guanine (G), leading to a amino acid change from Proline to Arginine in position 72. The five patients without any call for rs1042522 were classified as homozygous C/C, with a Pro72 phenotype. 19 patients with rs1042522 and a VF >99% were deemed as polymorphic G/G homozygotes, with an Arg72 phenotype. 11 patients in between were presumed to be heterozygous C/G-G/C, with a mixed phenotype.

3.2. TP53 Arg72Pro in Local Population

A comparison with the public repository Ensemble [5] from 1000GenomeProject was conducted. The healthy population from Tuscany (central Italy) held in this library, accounts for 6 homozygous G/G, 54 homozygous C/C and 47 heterozygous C/G-G/C [7]. The presence of the rs1042522 variant confers an OR of 6,11 (95%CI 2.20-16.94, p=.0005) towards pancreatic cancer when comparing our cohort of PaC patients to a healthy population coming from the same geographical area of Tuscany-central Italy (Table 2a). Moreover, if we consider only subjects deemed homozygous for G alleles in exon 4, locus 72, they present an OR of 20 (95%CI 6.93-57.61, p<.0001) towards pancreatic cancer (Table 2b).

4. Discussion

First discovered in 1986, p53 has two main species: one has an Arginine in position 72 that has a long positive charged side chain and the other, which has a Proline in the same position, has a small nonpolar side chain [8]. Rs1042522 is a single nucleotide variant (SNV) caused by the substitution of the wild type guanine (G) to cytosine (C) as in our findings, or adenosine (A) or thymine (T) [7]. According to the reference SNP (rs) report from the National Library of Medicine [9], G frequency ranges 0.11-0.495. 1000Genomes Project Phase 3 reports a frequency of G allele of 46% worldwide, being 0.276 in a Tuscan population. This European subpopulation accounts for 107 patients from central Italy, including the 6 patients with presented homozygous GG alleles in position 72 of TP53 gene [7].

Assuming that all patients from our cohort with a variant frequency >99% are likely homozygous G/G and that patients without any call for rs1042522 are C/C homozygous, our population had a C allele frequency of 0.3 and a G allele frequency of 0.7.

Regarding the Hardy Weinberg Equilibrium effect on case-control studies [10], according to which disequilibrium in the genetic variation of a population can be charged to different genetic background, inbreeding or genotyping problems, we believe that the multicentric enrollment along with the comparison with a public database overcome its effect on our results.

The Arg72 variant induces apoptosis five times better than the Pro72 variant, while the Pro72 represses the G1 phase of cell cycle [11,12]. This higher apoptotic potential is the mechanism thought to weaken the reserves of pancreatic beta cells leading to insulin depletion, causing diabetes. As a matter of fact, rs1042522 has been associated with increased odds of diabetes mellitus, [13] one of the conditions tightly linked to pancreatic cancer, both as a cause and as an early effect. New onset diabetic patients, within 3 years from diagnosis, have a prevalence of PaC of 1%, while long-standing type 2 DM patients have a RR of 1.8-2.1 for developing PaC [14]. If confirmed, the association of GG features to an increased risk of PaC could identify a very high risk population deserving to be screened.

Interestingly rs1042522 gives a susceptibility to gastric and esophageal cancer to Northwestern Chinese populations [15], as well to other cancer types such as gliomas, lung, bladder, prostate, colorectal and breast cancer [16]. On the other hand, a recent metanalysis showed that no association has been found between TP53 Arg72Pro and risk of ovarian cancer [17].

To the best of our knowledge, TP53 Pro72Arg polymorphism has been already described in 3 pancreatic cancer populations from Japan [18,19] and Czech Republic [20]. One study genotyped from FFPE tissues and showed an abnormal high C allele frequency of 0.8 [18]. The second and the latter genotyped from peripheral lymphocytes and have comparable C allele frequency of 0.29 - 0.39 [19,20]. In both cases it was hypothized a PaC higher risk in C allele carriers.

Despite our results in allele frequency are in line with those of Sonoyama [19] and Naccarati [21], we draw opposite conclusions for our population; in a similar way, p53 exon 4 (Pro) seems to be protective also for head and neck cancer in the Italian population [21].

Limits of this study are the lack of a matched control population as it compares a cohort of patients to a public database, moreover the small sample size along with the great variability of rs1042522 all over Italian population make the results not transposable on a larger scale, hence the need for a prospective large scale validation.

PaC is a major cause of death worldwide due to late diagnosis and the lack of screening programs. This results in a need to pinpoint high risk populations. TP53 is one of the four mountain genes responsible for PaC cancerogenesis and in our PaC population its polymorphism rs1042522 seemed to confer a 6 to 20 fold increased OR for PaC compared to a reference population. Further studies are needed to validate this hypothesis in order to improve patient selection for second level investigations and PaC early diagnosis.