Summary

METHODS
Thirty patients diagnosed with pancreatic cancer and 30 healthy controls who had no cancer history in their family and matched for age, gender, and ethnicity with the patients were analyzed in the study. The exome regions of the PALB2 gene in the genomic DNA obtained from the peripheral blood samples of the patients and controls were investigated by the Sanger sequencing method.

RESULTS
Evaluation of the obtained data showed that the alterations of c.29G>T, c.2737C>A, c.2773G>C, and c.2840T>G were only identified in the group of patient; and the alteration of c.1676A>G was detected in the homozygous formation in the case with a familial pancreatic cancer syndrome in the group of patient.

CONCLUSION
These alterations were suggested to be possibly important in the pathogenesis, and in the inheritance of pancreatic cancer. Further study is needed with a large cohort to emphasize importance of alteration.

Introduction

It is thought that genetic factors and genetic heterogeneity are some of the causes underlying the aggressive behavior of pancreas cancer.[2] Various germline mutations associated with pancreas cancer were detected in the literature. The most significant genes increasing the pancreas cancer risk were reported as BRCA1, BRCA2, PALB2, ATM, CDKN2A, APC, MLH1, MSH2, MSH6, PMS2, PRSS1 and STK11.[3-5]

Although the PALB2 gene was first described as associated with the Fanconi anemia, further studies also described the gene as the breast cancer susceptibility gene. The monoallelic truncating mutation of PALB2, which increased the breast cancer risk approximately 2-3 folds, was described in 10 out of 923 patients with a history of familial breast cancer.[6] PALB2 mutations were reported in approximately 3-4% of the families with familial pancreatic cancer syndrome.[7] In addition, the results of PALB2 gene analysis in BRCA negative families showed that the carriers of PALB2 mutation four folds increased the risk for male breast cancer, and six folds increased the risk for pancreas cancer.[8]

PALB2 is a gene that has a role in the DNA repair pathway by enabling the attachment and localization of BRCA2 protein to the BRCA complex. PALB2, which is the partner, and localizer of BRCA2 is emerged as the key player in the preservation of genome integrity. The monoallelic mutations in PALB2 are susceptible to breast, ovarian, and pancreatic cancer. The tumorsuppressor role of PALB2 is closely associated with its supporting ability of homologous recombination (HR) mediated repair of DNA double-strand breaks. Understanding the functioning of PALB2 has become the main focus point of various studies because PALB2 has been located at the intersection point of FA, HR, and cancer susceptibility.[9]

One of the repair mechanisms of homologous recombination was used in the repair of double-strand breaks during the transformation of S phase to G2 phase in the cell cycle, and that procedure was the main mechanism used in such repairs.[10,11] PALB2 gene product was suggested to have a tumor-suppressor role due to its interaction with the BRCA1 and BRCA2 proteins in double-strand DNA repair. The interaction of the BRCA complex with RAD51 enables the initiation of the homologous recombination. BRCA complex cannot establish an association with RAD51 in the absence of PALB2, and thus the repair of the double-strand DNA breaks cannot be performed.[12-15]

Special molecular alterations are known to be effective in the selection of personal treatment, such as platinum-based chemotherapy in pancreatic ductal adenocarcinoma (PDAC) patients. Although PALB2 was mainly investigated owing to its significant role in breast cancer biology, preliminary reports suggested that PALB2 might also have a role in pancreatic ductal adenocarcinoma. PDAC patients with PALB2 mutation were suggested to significantly benefit from the use of platinum-based chemotherapy based on a report of S. Boeck et al.[16] In addition, studies have reported that PDAC patients carrying a mutation in homologous recombination repair-related genes may better response to oxaliplatin-based chemotherapy.[17] Platinumbased agents directly target the DNA and cause DNA breaks through cross-links between the chains. PALB2 is suggested to be one of the potential biomarkers in genomic instability because PALB2 is an important mediator in DNA stability. Although platinum-based agents were found to be effective in pancreas cancer patients with genomic instability, the recent evidence showed that cancer cells might be resistant against platinum agents. The results of the studies reported that PARP inhibitor resistance was in the same clones where the second mutation developed.[18]

The investigation of the literature demonstrated that the distribution of the mutations in the PALB2 gene, which causes susceptibility to pancreas cancer, and breast cancer varied in accordance with the geographic regions.[19] The present study aims to identify the prevalence and distribution of PALB2 gene mutations in pancreas cancer patients in the Turkish population and investigate its role in the pathogenesis of the disease.

Methods

Mutation Analysis
The "Sanger sequencing" method was used to sequence full exons of the PALB2 gene in both healthy controls and patients with pancreas cancer. All exon regions and exon-intron boundaries of the PALB2 gene were amplified in the DNA samples of each patient and healthy controls through primers designed appropriately for the target gene using the PCR method. Polymerase chain reaction and sequencing procedures were performed for a total of 16 fragments consisting of four fragments in exon 4 and two fragments in exon 5.

The polymerase chain reaction was prepared as to provide a total final volume of 50 ?L consisting of 5 ?L from 10X PCR buffer, 1?L from 10 mM dNTP mixture, 0.5 ?L from each of the 0.3 mM primers, 2 ?L from the template DNA, 0.5 ?L from 5U/ ?L DreamTaq DNA polymerase, and 1 ?L MgCI2. The mixture was incubated for 20 seconds 94°C, 20 seconds at 56°C, and 40 seconds at 72°C in 35 cycles, and the final elongation was incubated 10 minutes at 72 °C.

The Roche HP PCR Product Purification kit was used for the purification of the PCR products. Thirteen exones of PALB2 gene were sequenced with a total of 16 amplicons for each case, and with 32 reactions using the DTCS Quick Start DNA Sequencing Kit. The exone sequencing after the precipitation procedures using ethanol was performed using "Beckman Coulter GXL" Sanger Sequencing Device, and the data were analyzed on the "GenomeLab Genetic Analysis System" program.

In Silico Analysis
The pathogenicity of the missense variants was evaluated in the databases of Polymorphism phenotyping (PolyPhen2),[20] the SIFT algorithms,[21] and Mutation Taster,[22] ClinVar [23] databases which are used in the identification of the pathologic possibilities.

Statistical Analysis
In this study, the non-parametric Chi-square (?²) test was used in the resolution of the data because our data (Kolmogorov-Smirnov Test: Assymp.Sig (significance): p<0.05) could not provide the normality assumption after conducting the single sample Kolmogorov- Smirnov test using the IBM Statistical Package for the Social Sciences (SPSS) v20.0 program.

Results

In this study, peripheral blood samples of 30 patients with pancreatic cancer applied to the Department of Basic Oncology, Faculty of Medicine, Istanbul University, were used. Peripheral blood samples of 30 healthy age and sex-matched individuals were used as a control group.

This study included 30 patients with pancreatic cancer, 13 of whom were women and 17 of whom were men along with 30 healthy individuals, of whom 20 men were examined. 27% (8/30) of the patients were under the age of 45 years, and 73% (22/30) of the patients were over the age of 45 years (Table 1). 10% (3/30) of the patients were in the 2nd stage, 47% (14/30) were in the 3rd stage, and 43% (13/30) were in the 4th phase (Table 1). While metastasis was detected in 83% (25/30) of the 30 patients, metastasis was not observed in 17% (5/30) (Table 1). 20% (6/30) of the patients had histological grade 1 (well-differentiated), 13% (4/30) grade 2 (moderate), and 17% (5/30) grade 3 (poorly differentiated). The histological grade of the tumor could not be evaluated in 50% (15/30) of the tumor (Table 1). While 60% (18/30) of the patients with pancreas had no familial history of cancer, 10% (3/30) had a family history of pancreatic cancer, and 30% (9/30) had at least one and four patients had another type of cancer history (Table 1).

Table 1: Clinical features of patients

When the patients compared according to another cancer or chronic disease, 43% (13/30) of the patients had a chronic disease, 57% (17/30) had no other chronic disease (Table 1). While 30% (9/30) of the patients examined in this study were diagnosed with diabetes, 70% (21/30) of the patients had no diagnosis of diabetes (Table 1). It was observed that 53% (16/30) of the patients used more than 20 years of smoking, and 47% (14/30) of the patients had no smoking during their lifetime.

The exons sequencing of the PALB2 gene was performed in 30 patients diagnosed with pancreas cancer, and the detected SNP alterations were compared with the results in the clinical database. The previously reported alterations of rs80531188, rs876659643, rs152451, rs45532440, rs180177125, and rs45551636, and 17 new, unreported, alterations in the literature were detected in this study (Table 2). Two of these alterations were reported as the VUS database, and the remaining four were shown as benign in the database of ClinVar Miner. In addition, 17 new SNP were found that they were not reported in the literature previously (Table 1 and Table 2). The chronograph images of alterations in DNA are indicated in Figure 1 and Figure 2, respectively (Fig. 1 and Fig. 2). In the study group, the alterations in the literature, which were determined for the first time, were evaluated using in silico algorithms, which were SIFT, Mutation Taster, PolyPhen 2. Two of these algorithms, which are used to determine the pathogenesis of a genetic change in scientific terms, are valid in the same direction. In this respect, the only change reported to be "pathogenic" in three of the three algorithms used in our study was c.29G>T. Changes in two of the three algorithms reported to be pathogenic "were c.2737C> A; c.2773G>C; c.2840T>G. Two of these changes are defined as a Variant of Uncertain Significance (VUS) in the ClinVar database and no literature information is available on the other 2 (Table 2).

Fig 1: The c.29G>T, rs876659643 polymorphism in the exon region 1 of the PALB2 gene in the DNA samples of the patient group. (a) SNP genotype and (b) normal genotype.

Fig 2: The c.1676A>G, rs152451 polymorphism in the exon region 4 of the PALB2 gene in the DNA samples of the patient group (reverse sequencing). (a) SNP genotype and (b) normal genotype.

Table 2: The detected alterations in the PALB2 gene of the patients with PC

When 30 healthy controls included in this study were sequenced for the PALB2 gene, heterozygous c.- 47G>A rs8053188, c.1676A>G rs152451, c.2014G>C rs45532440, c.2993G>A rs45551636 to the previously were reported as found changes (SNP) with the rs numbers. In addition, 13 different changes heterozygote formation in the same healthy group has not been previously reported in the literature were detected (Table 3), 1807C>G, c.2280T>G, c.2299G>A, c.2441T>A, c.2840T>G, c.3114-51A>T and c.3300T>G.

Table 3: The alterations in the PALB2 gene found only in the group of healthy control

The changes in the healthy control group without knowledge of the literature were calculated to be deleterious in two different in silico algorithms out of three, in two of them. Two changes were identified as benign in the ClinVar databank (Table 3).

Discussion

The c.29G>T, c.107A>G, and c.145A>G alterations were detected in a total of five patients out of 30 on the domain where the PALB2 protein interacts with BRCA1 protein and located in the N terminal region of the PALB2 protein. The alterations in this region suggested being effective in the formation of protein complex having a role in DNA repair and in the accurately functioning.

The alterations of c.559C>G, c.604C>G, c.611C>G, c.1005T>A, c.1027C>A, c.1368G>T, c.1391G>A, c.1464C>T, and c.1676A>G were recently detected in 19 patients (63%) out of 30 in the region including the domain three named as Chromatin-Association Motive (ChAM) located in the center of PALB2 protein, and reported to have a role in the chromatin localization of PALB2. Among these alterations, c.1676A>G alteration was also found with no rs152451, which was previously reported in the literature. This alteration was detected in five (17%) out of 30 patients and in four (13%) individuals in the control group. Four patients were detected to have an alteration in heterozygous formation, and one patient was detected to have an alteration in homozygous formation. The investigation of the clinical features of five patients having c.1676A>G alteration showed that tumor had distant organ metastasis, and all patients were smokers longer than 20 years. One patient was detected to have a pancreas cancer history in his father in whom homozygous c.1676A>G alteration was found. Therefore, particularly, the homozygous formation of the alteration was suggested to be important in the etiology and in the inheritance of the familial pancreas cancer. The importance of alteration could be detected with studies with a larger pancreatic cancer cohort and with a population- based group of healthy controls in the future.

All the alterations detected in our study were separately evaluated in the SIFT, Mutation Taster, and Polyphen algorithms that were developed to clarify the differences caused in the protein structures and to identify how they affected the protein function. These three algorithms, named as SIFT, Mutation Taster, and Polyphen, are used in the identification of the pathogenicity of scientifically genetic alteration. The conditions where the two of these algorithms give results in the same direction are regarded valid. Considering this, the only alteration reported as "deleterious" was c.29G>T in all three algorithms in our study. The alterations regarded as "deleterious" in two out of three algorithms were the alterations c.2737C>A; c.2773G>C; and c.2840T>G.

The alterations of c.29G>T, c.2737C>A, c.2773G>C, and c.2840T>G detected in the patient group, and were calculated as "deleterious" in at least two algorithms could not be shown in any cases in the control group. The results suggested that these alterations might be important in the pancreas cancer pathogenesis. In addition, c.1676A>G alteration was observed in 17% of the patient group and in 13% of the healthy individuals in our study group. The c.1676A>G alteration was detected in heterozygous formation except for one patient with pancreas cancer syndrome in the patient group. The heterozygous formation of this alteration was reported as "pathogenic" in accordance with the ClinVar data bank. However, the detection of the homozygous formation of c.1676A>G alteration in a patient with a family history of pancreas cancer in our study group suggested that the homozygous alteration might be important for the pancreas cancer pathogenesis, and was a topic to be investigated. In addition, the condition in the patient in whom the homozygous c.1676A>G alteration was detected in the first-degree relative demonstrated a similar condition to SMAD4/DPC4 gene which is known to be effective in the pancreas cancer etiology, and which showed a homozygous mutation in 50% of the patients with pancreas cancer. The detection of the heterozygous formation of this alteration in the same ratio in both control, and patient groups, and detection of homozygous formation particularly in a patient with a family history of pancreas cancer in the first degree relative suggested that the homozygous formation of that region might be important in the pancreas cancer etiology similar to the homozygous form of the SMAD4/DPC4 gene. The investigation of this condition, particularly in pancreas cancer tissue samples in future studies, will provide information about this alteration.

Conclusion

Peer-review: Externally peer-reviewed.

Conflict of Interest: The authors declare that they have no competing interests.

Ethics Committee Approval: The material collection processes of all patients and controls were approved by the Istanbul University Ethics Board (protocol number: 2014/1961).

Financial Support: This work was supported by the Scientific Research Projects Coordination Unit of Istanbul University (project number: 54273).

Authorship contributions: Concept - G.K.T., D.A.Ö., M.A., Ş.B.T., S.K.E., Ö.Ş.E., D.T., S.K., E.K., H.Y.; Design - G.K.T., D.A.Ö., M.A., Ş.B.T., S.K.E., Ö.Ş.E., D.T., S.K., E.K., H.Y.; Supervision - G.K.T., D.A.Ö., M.A., Ş.B.T., S.K.E., Ö.Ş.E., D.T., S.K., E.K., H.Y.; Funding ? G.K.T., H.Y.; Materials ? G.K.T., D.A.Ö., M.A., Ş.B.T., S.K.E., Ö.Ş.E., D.T., S.K., E.K., H.Y.; Data collection &/or processing -G.K.T., D.A.Ö., M.A., Ş.B.T., S.K.E., Ö.Ş.E., D.T., S.K., E.K., H.Y.; Analysis and/or interpretation - G.K.T., D.A.Ö., M.A., Ş.B.T., S.K.E., Ö.Ş.E., H.Y.; Literature search - G.K.T., D.A.Ö., M.A., Ş.B.T., S.K.E., Ö.Ş.E., H.Y.; Writing - G.K.T., H.Y.; Critical review - G.K.T., D.A.Ö., M.A., Ş.B.T., S.K.E., Ö.Ş.E., D.T., S.K., E.K., H.Y.

References

1) Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68(6):394-424.

2) Matthaios D, Zarogoulidis P, Balgouranidou I, Chatzaki E, Kakolyris S. Molecular pathogenesis of pancreatic cancer and clinical perspectives. Oncology 2011;81(3-4):259-72.

3) Solomon S, Das S, Brand R, Whitcomb DC. Inherited pancreatic cancer syndromes. Cancer J 2012;18(6):485-91.

4) Zhan W, Shelton CA, Greer PJ1, Brand RE, Whitcomb DC. Germline Variants and Risk for Pancreatic Cancer: A Systematic Review and Emerging Concepts. Pancreas 2018;47(8):924-36.

5) Chaffee KG, Oberg AL, McWilliams RR, Majithia N, Allen BA, Kidd J, et al. Prevalence of germ-line mutations in cancer genes among pancreatic cancer patients with a positive family history. Genet Med 2018;20(1):119-27.

6) Rahman N, Seal S, Thompson D, Kelly P, Renwick A, Elliott A, et al; Breast Cancer Susceptibility Collaboration (UK). PALB2, which encodes a BRCA2-interacting protein, is a breast cancer susceptibility gene. Nat Genet 2007;39(2):165-7.

7) Jones S, Hruban RH, Kamiyama M, Borges M, Zhang X, Parsons DW, et al. Exomic sequencing identifies PALB2 as a pancreatic cancer susceptibility gene. Science 2009;324(5924):217.

8) Casadei S, Norquist BM, Walsh T, Stray S, Mandell JB, Lee MK, et al. Contribution of inherited mutations in the BRCA2-interacting protein PALB2 to familial breast cancer. Cancer Res 201115;71(6):2222-9.

9) Pauty J, Rodrigue A, Couturier A, Buisson R, Masson JY. Exploring the roles of PALB2 at the crossroads of DNA repair and cancer. Biochem J 2014;460(3):331-42.

10) Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with knives. Mol Cell 2010;40(2):179-204.

11) Moynahan ME, Jasin M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat Rev Mol Cell Biol 2010;11(3):196-207.

12) Sy SM, Huen MS, Chen J. PALB2 is an integral component of the BRCA complex required for homologous recombination repair. Proc Natl Acad Sci U S A 2009;106(17):7155-60.

13) Zhang F, Fan Q, Ren K, Andreassen PR. PALB2 functionally connects the breast cancer susceptibility proteins BRCA1 and BRCA2. Mol Cancer Res 2009 Jul;7(7):1110-8.

14) Zhang F, Ma J, Wu J, Ye L, Cai H, Xia B, et al. PALB2 links BRCA1 and BRCA2 in the DNA-damage response. Curr Biol 2009;19(6):524-9.

15) Tischkowitz M, Xia B. PALB2/FANCN: recombining cancer and Fanconi anemia. Cancer Res 2010;70(19):7353-9.

16) Boeck S, Mehraein Y, Ormanns S, Kruger S, Westphalen CB, Haas M, et al. Mismatch-repair-deficient metastatic pancreatic ductal adenocarcinoma with a germline PALB2 mutation: unusual genetics, unusual clinical course. Ann Oncol 2017;28(2):438-9.

17) Kondo T, Kanai M, Kou T, Sakuma T, Mochizuki H, Kamada M, et al. Association between homologous recombination repair gene mutations and response to oxaliplatin in pancreatic cancer. Oncotarget 2018;9(28):19817-25.

18) Sahin IH, Lowery MA, Stadler ZK, Salo-Mullen E, Iacobuzio-Donahue CA, Kelsen DP, et al. Genomic instability in pancreatic adenocarcinoma: a new step towards precision medicine and novel therapeutic approaches. Expert Rev Gastroenterol Hepatol 2016;10(8):893-905.

19) Borecka M, Zemankova P, Vocka M, Soucek P, Soukupova J, Kleiblova P, et al. Mutation analysis of the PALB2 gene in unselected pancreatic cancer patients in the Czech Republic. Cancer Genet 2016;209(5):199-204.

20) Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Methods 2010;7(4):248-9.

21) Kumar P, Henikoff S, Ng PC. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc 2009;4(7):1073-81.

22) Schwarz JM, Cooper DN, Schuelke M, Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods 2014;11(4):361-2.

23) Silvestri V, Barrowdale D, Mulligan AM, Neuhausen SL, Fox S, Karlan BY, et al. Male breast cancer in BRCA1 and BRCA2 mutation carriers: pathology data from the Consortium of Investigators of Modifiers of BRCA1/2. Breast Cancer Res 2016;18(1):15.

24) Hirsch JA, James P, Robinson JR, Eastman KM, Conley KD, Evenson KR, et al. Using MapMyFitness to Place Physical Activity into Neighborhood Context. Front Public Health 2014;2:19.