EBioMedicine 53 (2020) 102675 Contents lists available at ScienceDirect EBioMedicine journal homepage: www.elsevier.com/locate/ebiom Research paper A comprehensive analysis of candidate genes in familial pancreatic cancer families reveals a high frequency of potentially pathogenic germline variants Julie Earla,b,*, Cristina Galindo-Pumarin ~ oa,b, Jessica Encinasa, Emma Barretoa, Maria E. Castilloa, Vanessa Pacho na,b, Reyes Ferreiroa, Mercedes Rodríguez-Garrotea,b, Silvia Gonza lez-Martíneza, Teresa Ramon y Cajalc, Luis Robles Diazd, Isabel Chirivella-Gonzaleze, Montse Rodriguezf, Eva Martínez de Castrog, David García-Seisdedosh, Gloria Mun ~ ozh, Juan Manuel Rosa Rosai, Mirari Marquezj,b, j,b a,b Nuría Malats , Alfredo Carrato a Molecular Epidemiology and Predictive Tumor Markers Group, Medical Oncology Research Laboratory, Ramo n y Cajal Health Research Institute (IRYCIS), Carretera Colmenar Km 9100, 28034 Madrid, Spain b Biomedical Research Network in Cancer (CIBERONC), C/Monforte de Lemos 3-5, Pabello n 11, 28029 Madrid, Spain c Medical Oncology Department, Santa Creu i Sant Pau Hospital, Mas Casanovas, 90, 08041 Barcelona, Spain d Familial and Hereditary Cancer Unit. Medical Oncology Department, 12 de Octubre Hospital, Av. Cordoba, s/n, 28041 Madrid, Spain e n Genetic Counselling Unit, Valencia University Hospital Clinic, Av. de Blasco Iba ~ ez, 17, 46010 Valencia, Spain f ~ a Biomedical Research Institute, Hospital Teresa Herrera, Xubias de Arriba, 84, 15006 A Corun A Corun ~ a, Spain g Medical Oncology Department, Marques de Valdecilla University Hospital, Av. Valdecilla, 25, 39008 Santander, Spain h Translational Genomics Core Facility, Instituto Ramon y Cajal de Investigacion Sanitaria (IRYCIS), Madrid, Spain i Pathology Department, Instituto Ramo n y Cajal de Investigacion Sanitaria (IRYCIS), Madrid, Spain j Genetic and Molecular Epidemiology Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain A R T I C L E I N F O A B S T R A C T Article History: Background: The 5-year survival rate of patients with pancreatic ductal adenocarcinoma (PDAC) is around 5% due Received 30 September 2019 to the fact that the majority of patients present with advanced disease that is treatment resistant. Familial pancreatic Revised 29 January 2020 cancer (FPC) is a rare disorder that is defined as a family with at least two affected first degree relatives, with an esti- Accepted 29 January 2020 mated incidence of 4% 10%. The genetic basis is unknown in the majority of families although around 10% 13% of Available online 27 February 2020 families carry germline mutations in known genes associated with hereditary cancer and pancreatitis syndromes. Methods: Panel sequencing was performed of 35 genes associated with hereditary cancer in 43 PDAC cases from Keywords: families with an apparent hereditary pancreatic cancer syndrome. Findings: Pathogenic variants were identified in Familial pancreatic cancer Panel sequencing 19% (5/26) of PDAC cases from pure FPC families in the genes MLH1, CDKN2A, POLQ and FANCM. Low frequency DNA repair and hereditary cancer genes potentially pathogenic VUS were also identified in 35% (9/26) of PDAC cases from FPC families in the genes FANCC, Pathogenic variants MLH1, PMS2, CFTR, APC and MUTYH. Furthermore, an important proportion of PDAC cases harboured more than one pathogenic, likely pathogenic or potentially pathogenic VUS, highlighting the multigene phenotype of FPC. Interpretation: The genetic basis of familial or hereditary pancreatic cancer can be explained in 21% of families by previously described hereditary cancer genes. Low frequency variants in other DNA repair genes are also present in 35% of families which may contribute to the risk of pancreatic cancer development. Funding: This study was funded by the Instituto de Salud Carlos III (Plan Estatal de I + D + i 2013 2016): ISCIII (PI09/02221, PI12/01635, PI15/02101 and PI18/1034) and co-financed by the European Development Regional Fund ‘‘A way to achieve Europe’’ (ERDF), the Biomedical Research Network in Cancer: CIBERONC (CB16/12/00446), Red Tema tica de investigacio  n cooperativa en ca ncer: RTICC (RD12/0036/0073) and La Asociacio  n Espan~ ola contra el Ca ncer: AECC (Grupos Coordinados Estables 2016). © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/) * Corresponding author at: Molecular Epidemiology and Predictive Tumor Markers Group, Medical Oncology Research Laboratory, Ramo  n y Cajal Health Research Insti- tute (IRYCIS), Carretera Colmenar Km 9100, 28034 Madrid, Spain. E-mail address: julie.earl@salud.madrid.org (J. Earl). https://doi.org/10.1016/j.ebiom.2020.102675 2352-3964/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/) 2 J. Earl et al. / EBioMedicine 53 (2020) 102675 has identified germline mutations in 6 genes including CDKN2A, Research in context TP53, MLH1, BRCA2, ATM and BRCA1 that are associated with pancre- atic cancer development in 5.5% of all pancreatic cancer (PC) cases, Evidence before this study both sporadic and those with a family history of pancreatic cancer Previous studies have identified germline mutations in [14,15]. FPC appears to be inherited in an autosomal dominant man- CDKN2A, TP53, MLH1, BRCA2, ATM and BRCA1 via next-genera- ner and around 10% 13% of families carry germline mutations in tion sequencing that are associated with pancreatic cancer BRCA2, PALB2, ATM, CHEK2, CDKN2A, Lynch syndrome mismatch development in an estimated 5.5% of unselected PDAC cases. repair genes, Fanconi anaemia related genes and PRSS1 and SPINK2 Approximately 10% 13% of FPC families carry germline muta- (hereditary pancreatitis) as well as other novel genes [16,17,18]. tions in BRCA2, PALB2, ATM, CHEK2, CDKN2A, Lynch syndrome However, the major underlying genetic defect(s) are still unknown in mismatch repair genes, Fanconi anaemia related genes and the majority of families. Families present with either a high incidence PRSS1 and SPINK2 (hereditary pancreatitis), among others. A of only pancreatic cancer or in combination with other cancer syn- search of the current literature was performed to identify genes dromes such as Hereditary Breast and Ovarian Cancer (HBOC), Peutz associated with hereditary pancreatic cancer risk and a panel of Jeghers Syndrome (PJS) and Familial Atypical Multiple Mole Mela- 35 genes was selected to perform a genetic screening of familial noma (FAMMM). PDAC cases. One of the keys to improving patient prognosis is early diagnosis and FPC is a known high risk population that would benefit from Added value of this study early detection strategies. The Spanish familial pancreatic cancer reg- istry (Pan-Gen-FAM) was established in 2009 in Spain with the prin- On the whole, this study provides increasing evidence that the cipal objective to characterise the phenotypic and genetic genetic basis of hereditary pancreatic cancer can be explained by background of FPC [19]. The registry currently includes over 200 indi- previously described hereditary cancer associated genes such as viduals representing some 88 families presenting with pancreatic MLH1, CDKN2A, POLQ and FANCM. In addition, previously unde- cancer alone or in combination with known cancer syndromes. scribed likely pathogenic variants were found in PTEN, POLQ and Healthy high risk individuals are offered a secondary screening pro- TET2 in PDAC cases from 2 different families, which may be novel gram for the early detection of PC at a potentially curative stage. This variants associated with this syndrome. Furthermore, low fre- consists of annual magnetic resonance imaging (MRI) and endoscopic quency potentially pathogenic VUS were identified in additional ultra-sound (EUS) and blood biomarkers, including CA19-9 analysis genes such as FANCC, PMS2, CFTR, APC and MUTYH, although their in serum [20,21]. role in pancreatic cancer risk needs to be clarified in other cohorts. The aim of the study was to analyse individuals recruited to the familial pancreatic registry for a pathogenic germline mutation in Implications of all the available evidence genes frequently associated with hereditary cancer. Panel sequencing The understanding of the genetic basis of hereditary pancreatic was performed of 35 genes associated with hereditary cancer in 43 cancer has important implications for the identification of true PDAC cases with an apparent hereditary pancreatic cancer syndrome, high risk individuals in order to optimise secondary screening 11 high risk individuals who participate in the early detection pro- strategies. gram and 9 individuals diagnosed with non-pancreatic tumours or pancreatitis from families within the Pan-Gen-FAM registry. 2. Methods 1. Introduction 2.1. Recruitment of cases and high risk individuals The prognosis of patients diagnosed with pancreatic ductal adenocar- cinoma (PDAC) is dismal with a 5-year survival rate of around 5% as the This study was approved by the local ethics committee. High risk majority of patients present with advanced disease, which is aggressive families were identified in the clinical oncology or familial cancer and treatment resistant. Screening high-risk populations would result in unit in 10 hospitals participating throughout Spain. High risk families an earlier diagnosis and a higher chance for cure. Very few risk factors with the following phenotype or characteristics were included in the have been identified, although there is good evidence to suggest that study: Familial pancreatic cancer families with at 2 affected first smoking, obesity, a family history of pancreatic cancer, pancreatitis and degree relatives (FPC), HBOC families with at least one case of PC, diabetes increase pancreatic cancer risk [1]. More recently, it has been FAMMM families, Hereditary Non Polyposis Colorectal Cancer shown that alterations in the microbioma may play a role in PC develop- (HNPCC) families with at least one case of PC and PC cases diagnosed ment [2]. There are no effective biomarkers for early detection in the at 50 years of age. Patients were asked to sign the informed consent general population. CA19-9 is currently used in the clinic, although the before inclusion into the study and an exhaustive family tree consist- sensitivity and specificity for the diagnosis of symptomatic pancreatic ing of at least 3 generations was prepared for each participating fam- cancer is 79% 81% and 82% 90%, respectively [3]. Several potential bio- ily. The index case reported the occurrence of pancreatic cancer and markers have been recently described for early detection, such as a other relevant cancer types and diseases within the family. Blood three-protein panel in urine [4], Galectin-1 (Gal-1) in serum [5], throm- samples in EDTA tubes were taken on entry into the study and leuco- bospondin-2 (THBS2) in plasma [6], circulating tumour DNA (ctDNA) [7] cytes and lymphocytes fractions were isolated from whole blood and and the IMMrayTM PanCan-d 29 biomarker signature in serum [8]. stored until DNA extraction and sequencing analysis. All clinical and Sporadic PDAC occurs worldwide at an approximate frequency of personal data was stored in a secure database (RedCap). 7 in 100,000 person-years and its incidence rate increases at 1% per year [9,10]. However, the risk of developing PDAC increases accord- 2.2. Next generation sequencing of PDAC cases, high risk individuals and ing to the number of affected family members, the standard inci- non-PDAC cases dence ratio is 4.6 with one affected family member to 32 with three affected family members [11]. Familial pancreatic cancer (FPC) is A power calculation was performed using the online tool ClinCalc. defined as a family with at least one pair of affected first degree rela- com (https://clincalc.com/stats/samplesize.aspx). A group of 38 96 tives and an estimated 4% 10% of pancreatic cancers diagnosed have PDAC cases were required to identify pathogenic variants with an a familial background [12,13]. Next-generation sequencing analysis assumed frequency of 1% 2% in the test population (PDAC familial J. Earl et al. / EBioMedicine 53 (2020) 102675 3 cases) and 0.001% 0.005% in the general population, with a power of the 64 selected variants according to the different databases con- 0.8, alpha value of 0.05 and a Beta value of 0.2. All diagnosed PDAC sulted is included in Supplementary Table 1. Variants were finally cases with a source of germline DNA (lymphocytes or leucocytes) classified into 3 groups. (1) Pathogenic variants, which were missense were included in the sequencing study and finally 43 cases were or premature stop variants with a demonstrated pathogenic potential included. According to the power calculation, this would allow patho- in familial pancreatic cancer or other familial cancer syndromes. (2) genic mutations to be identified with an assumed 2% frequency in the Likely Pathogenic variants, which were premature stop variants with test population and 0.005% frequency in the general population, with an unknown role in familial pancreatic cancer. (3) Potentially patho- a power of 0.8, alpha value of 0.05 and a Beta value of 0.2. Briefly, genic variants with an unknown significance (VUS), which were genomic DNA was extracted from leucocyte using the Flexigene DNA defined as missense or splice site variants with an unknown signifi- kit (Qiagen) according to the manufacturers instructions and quanti- cance in familial pancreatic cancer. fied using the Qubit V2.0 Fluorometer (Life Technologies). A search of the literature was performed using the terms “familial pancreatic 2.4. Validation of pathogenic and likely pathogenic variants cancer” and “hereditary pancreatic cancer” in order to identify publications that report germline mutations associated with pancre- The corresponding genomic sequence of the pathogenic and likely atic cancer. Furthermore, a search for “pancreatic cancer gene panels” pathogenic variants was retrieved from the ensemble Human was performed in order to identify additional genes that are included GRCh38.p12 data base (https://www.ensembl.org/index.html). Pre- in commercially available sequencing panels. A targeted capture designed PCR/Sanger Sequencing primers (ThermoFisher) were used sequencing panel that included 35 genes associated with cancer was for validation when available. If not, primers were designed using the created. The genes included in the panel were APC, ATM, BMPR1A, primer blast option in the NCBI database (https://www.ncbi.nlm.nih. BRCA1, BRCA2, CDH1, CDKN2A, CHEK2, EPCAM, MLH1, MSH2, MSH6, gov/tools/primer-blast/) and PCR amplicons from 400 to 800 bp were MUTYH, PALB2, PMS2, PTEN, SMAD4, STK11, TP53, VHL, PRSS1, TERT, selected. DNA was isolated from an independent leucocyte or lym- CFTR, TET2 9, DNMT3A, POLN 6, POLQ 6, ASXL1 5, FANCG 4, BUB1B 3, phocyte sample from the same patient using the DNA and Blood DNA ESCO2 3, FANCC 3, FANCM 3, MSH4 3 and RAD54L. The panel was isolation kit (Qiagen). DNA was quantified by nanodrop and designed using the Agilent SureDesign Software (Agilent Technolo- 10 30 ng of genomic DNA was used for each PCR reaction. PCR was gies Inc.). 50 ng of genomic DNA was fragmented and adaptors were performed using the Amplitaq gold reagent (Applied Biosystems) added in a single enzymatic step. The adaptor-tagged DNA library according to the manufacturer’s instructions. The PCR conditions of was purified and amplified. Next, 750 ng of each library were hybri- each primer pair was optimised on an individual basis and the list of dised using SureSelectQXT capture library for 90 min. The resulting primers used and corresponding PCR conditions are shown in Sup- libraries were recovered using streptavidin magnetics beads, and a plementary Table M1. Sanger sequencing was performed using the post-capture PCR amplification was carried out. Library fragment size Big Dye Terminator kit (Applied Biosystems) and fragments were distribution was assessed using the 2200 TapeStation Instrument analysed using the ABI prism system at the Complutense University (Agilent Technologies). Sequencing was performed on the MiSeq of Madrid. Sequences were analysed manually by visualization of the (Illumina) using paired-end protocol 2 £ 150 bp and Micro V2 chem- chromatograms and using the Blast suite of programs in the NCBI istry. On average, 5.1 million of pass filter reads were produced per database (https://www.ncbi.nlm.nih.gov/). sequencing run and 93.9% of reads had an average Phred score qual- ity above Q30 and the read depth was greater than 200. Samples 2.5. Statistical analysis from the trio family CEPH1463 [22] were used to validate the sequencing panel and determine the sensitivity, specificity and over- The Chi-square or Fishers exact test were used to analyse the dif- all depth of the sequencing panel. Further information is available in ferences in the frequency of the different variant types between the 3 supplementary methods. patient groups (PDAC cases, non-PDAC cases and high risk individu- als) and the different family phenotypes (FPC vs. HBOC-PC). 2.3. Identification of potentially pathogenic mutations 3. Results The Ingenuity variant analysis (IVA) software was used to analyse the sequencing data and identify pathogenic and potentially patho- 3.1. Validation of the sequencing panel genic variants. The tertiary analysis strategy is summarised in Sup- plementary methods and Supplementary Fig. M1. Briefly, “Genetic Sequencing was performed in a CEPH trio reference panel (two Disease” was selected as the analysis design and the biological term parents and their child) of European origin (www.hapmap.org) [22], the “familial cancer” (germinal disease) was used as the primary filter. sensitivity of the panel was 0.995475 and the specificity was 0.996492. The variants identified were filtered based on the following criteria. With regard to the panel design used in this study, 98.53% of the panel (1) An allele fraction  25 and call quality  20 was used to exclude had a depth of greater than 200X and the coverage at this depth was false positives, only variants that complied with this criteria in both always greater than 98%. Panel sequencing was also performed in 3 pre- cases and controls (CEPH trio) were retained. (2) Common variants viously identified BRCA2 mutation carriers, one individual with a BRCA2 with a frequency of more than 1% in the general population were and BRCA1 germline mutation one CDKN2 mutation carrier from the excluded. (3) Variants with a predicted pathogenic or likely patho- Pan-Gen-FAM registry in order to validate the panel for the ability to genic consequence according to the ACMG guidelines were retained, detect pathogenic mutations. All mutations were successfully identified which included nonsense (STOP gain), frameshift, missense with a with the panel (Supplementary Tables 1 and 2). damaging or possibly damaging effect according to SIFT or PolyPhen- 2 function predictions and canonical splice site variants. (4) Variants 3.2. Identification of pathogenic, likely pathogenic variants and with an inferred gain or loss of function were selected in the final potentially pathogenic VUS in PDAC cases “genetic analysis” parameter. The reported pathogenic potential of these 64 variants in hereditary cancer syndromes was assessed in Panel sequencing was performed in 43 PDAC cases from 43 different various databases including ClinVar (https://www.ncbi.nlm.nih.gov/ families, one case was also previously diagnosed with breast cancer. The clinvar/), InSiGHT (https://www.insight-group.org/syndromes/lynch- characteristics of these cases are shown in Table 1 and Supplementary syndrome/) and HGMD: The Human Gene Mutation Database (http:// Table 1. Pathogenic, likely pathogenic or potentially pathogenic VUS www.hgmd.cf.ac.uk/ac/index.php). The predicted pathogenicity of were identified in 32/43 (74%) of PDAC cases and 11/43 (26%) cases 4 J. Earl et al. / EBioMedicine 53 (2020) 102675 Table 1 Table 2 Characteristics of the 43 PDAC cases included in the study. Pathogenic and likely pathogenic variants identified in PDAC cases from familial pan- creatic cancer families. Demographic data Family Genbank accession Variant classification Sex 19 males 24 females Phenotype and transcript Median age (range) 59.5 (29 84) Median age males (range) 57 (31 80) FPC NM_000077.4 (CDKN2A): Pathogenic variants with Median age female (range) 62 (29 84) c.176T>G (p.Val59Gly) a determined pathogenic Family phenotype FPC NM_000249.3(MLH1): role in familial cancer FPC 26 c.1852A>G (p.Lys618Glu) HBOC + PC 8 HBOC + PC NM_000249.3(MLH1): CYSTIC FIBROSIS + PC 1 c.1852A>G (p.Lys618Glu) PC<=50 years 8 FPC NM_199,420.3(POLQ): Likely pathogenic premature FPC: at least 2 affected 1st degree relatives; HBOC + PC: hereditary breast and ovarian c.4541_4544delCTTC stop variants with an unknown cancer with at least one case of PC; HBOC + PC <=50 Y: hereditary breast and ovarian (p.P1514fs*3) consequence in familial cancer with at least one case of PC less than 50 years of age; PC<=50 years: 1 affected HBOC + PC NM_007194.3(CHEK2): pancreatic cancer case less than 50 years of age; FAMMM-PC: FAMMM syndrome with at least one case c.409C>T (p.Arg137Ter) of PC. CYSTIC FIBROSIS + PC: case of cystic fibrosis and pancreatic cancer. PC<=50 years NM_000314.6(PTEN): c.156delG (p.H53fs*9) were negative for variants. These were most frequently found in the HBOC + PC NM_001127208.2 (TET2): genes MLH1, CFTR, FANCC and RAD54L and the frequency of these var- c.1954C>T (p.Q652*) and c.3368delC iants is shown in Supplementary Table 1 and Fig. 1. 9/43 (21%) cases (p.P1123fs*14) had pathogenic or likely pathogenic variants and 3 of these cases also FPC NM_199,420.3(POLQ): had potentially pathogenic VUS. Pathogenic and likely pathogenic var- c.7486dupC (p.H2496fs*17) iants were validated by Sanger sequencing in the original DNA sample FPC NM_020937.3(FANCM): c.5791C>T (p.Arg1931Ter) used for panel sequencing as well as an independent sample of lympho- cytes from the same patient and the results were consistent. FPC: at least 2 affected 1st degree relatives; FPC + <=50 years: 2 affected 1st degree rela- Pathogenic variants were found in 3/43 (7%) cases, which involved the tives and one diagnosed at less than age 50; HBOC + PC: hereditary breast and ovarian cancer with at least one case of PC; HBOC + PC <=50 Y: hereditary breast and ovarian can- genes MLH1 and CDKN2A. The pathogenic MLH1 variant c.1852A>G (p. cer with at least one case of PC less than 50 years of age; PC<=50 years: 1 affected case Lys618Glu) was found in 2 cases from 2 different families and the less than 50 years of age; FAMMM-PC: FAMMM syndrome with at least one case of PC. CDKN2A c.176T>G (p.Val59Gly) variant was found in one case (Table 2). Likely pathogenic variants (premature stop codon) with an unknown role in FPC were also identified in 6/43 (14%) cases involving the POLQ, CHEK2, PTEN, TET2 and FANCM genes (Table 2). The variants in the genes VUS. 8 potentially pathogenic VUS were identifie d in 2 or more cases FANCM, POLQ and CHEK2 were identified in 3 individuals from different from different families (Supplementary Table 1). These variants all have families and have been previously described in public databases. Previ- a MAF of  0.01. The FANCC variant c.584A>T (p.Asp195Val) was found ously undescribed variants in PTEN, and POLQ were also identified in 2 in 4 PDAC cases from 4 different families and the RAD54L variant cases from 2 different families. Two likely pathogenic variants were iden- c.1487G>C (p.G496A) was found in 2 PDAC and one breast case from 3 tified in TET2 in the same individual, one of which had been previously different families. Potentially pathogenic VUS in PMS2 (c.52A>G (p. described. Pathogenic and likely pathogenic variants were most fre- Ile18Val)), MLH1 (c.1853A>C (p.Lys618Trp)) and MUTYH (c.1187G>A quently found in the genes PTEN, CDKN2A, FANCM and POLQ in FPC fami- (p.Gly396Asp)) were found in 2 PDAC cases from 2 different families lies. Whereas, pathogenic and likely pathogenic variants in CHEK2 and and the c.3949G>C (p.Glu1317Gln) variant in APC was found in one TET2 were most frequently found in HBOC+PC families (Fig. 2a). Variants PDAC case and 1 breast case from 2 different families (Table 3). in MLH1 were found in both FPC and HBOC- PC families (Fig. 2a). The dif- Potentially pathogenic VUS were most frequently found in the ference in the frequency of variants between the different family pheno- genes CFTR, MSH2, FANCC and PMS2 in FPC families. Whereas, variants types was not statistically significant according to the Fishers exact test. in MUTYH, TET2 and CHEK2 were most frequently found in HBOC+PC Potentially pathogenic VUS missense or splice site variants were families (Fig. 2b). The difference in the frequency of variants between found in 26/43 (60%) PDAC cases. 23/43 (53%) had only potentially the different family phenotypes was not statistically significant pathogenic VUS and 8/43 (19%) had 2 or more potentially pathogenic according to the Fishers exact test. Fig. 1. The frequency of pathogenic and potentially pathogenic VUS in 43 sequenced cases. J. Earl et al. / EBioMedicine 53 (2020) 102675 5 Fig. 2a. The frequency of pathogenic and likely pathogenic variants in PDAC cases from FFC and HBOC+PC families. Fig. 2b. The frequency of potentially pathogenic VUS in PDAC cases from FFC and HBOC+PC families. Table 3 Potentially pathogenic VUS identified in 2 or more individuals/PDAC cases from different families. Genbank accession an transcript rs code MAF Frequency Number of cases/individuals and family phenotype (dbSNP) NM_000136.2(FANCC):c.584A>T (p.Asp195Val)* 1800365 0.011 4 PC cases (3 from FPC family and 1 from HBOC + PC family) NM_001142548.1(RAD54L):c.1487G>C (p.G496A)* 138546115 0.002 2 PC cases and 1 PC/breast case (PC case from HBOC + PC and PC/breast case from FPC and PC case from PC<=50 years family) NM_000535.6(PMS2):c.52A>G (p.Ile18Val)* 63750123 0.01 2 PC cases (1 from FPC and 1 from FPC>=3 PC family) 0NM_000249.3(MLH1):c.1853A>C (p.Lys618Trp)* 63750449 0.001 2 PC cases (1 from PC<=50 years and 1 from FPC>=3 PC family) NM_001128425.1 (MUTYH): c.1187G>A (p.Gly396Asp)* 36053993 0.009 2 PC cases (1 from FPC and1 from HBOC + PC family) NM_000038.5(APC):c.3949G>C (p.Glu1317Gln) 1801166 0.006 1 PC case and 1 breast case (PC case from FPC and breast case from HBOC + PC family) NM_000492.3(CFTR):c.2758G>T (p.Val920Leu) 373885282 0.00004 1 PC cases and 2 high risk (1 PC case from FPC and 2 high risk from the same FPC>=3 PC family) NM_001211.5(BUB1):c.341G>A (p.Arg114Gln) 1,372115983 0.000008 1 high risk and 1 BRCA2 carrier from HBOC + PC <=50 Y families * variants found in at least 2 PC cases from different families. FPC: at least 2 affected 1st degree relatives; FPC>=3 PC: 3 or more affected 1st degree relatives; FPC + <=50 years: 2 affected 1st degree relatives and one diagnosed at less than age 50; HBOC + PC: hereditary breast and ovarian cancer with at least one case of PC; HBOC + PC <=50 Y: hereditary breast and ovarian cancer with at least one case of PC less than 50 years of age; PC<=50 years: 1 affected case less than 50 years of age. 3.3. Potentially pathogenic VUS in cases with other cancer types and high tumour, 1 colon cancer, 1 liver cancer, 1 thyroid cancer, 1 ampuloma risk individuals and 1 pancreatitis case. Sequencing was also performed in 11 high risk individuals from 9 different families, who participate in the early Panel sequencing was also performed in 9 individuals diagnosed detection program. 8 of these high risk individuals were diagnosed with other tumour types or pancreatitis from 9 additional families, with a relevant lesions during follow-up, this included 2 suspected this included 3 breast cancer cases, 1 pancreatic neuroendocrine PDAC cases and 1 pancreatic neuroendocrine tumour (pNET) case 6 J. Earl et al. / EBioMedicine 53 (2020) 102675 that underwent a surgical resection, 2 IPMN and 3 pancreatic cysts. Pathogenic, likely pathogenic and potentially pathogenic VUS) The characteristics of the cases/high risk individuals and their family were found in MLH1, TET2, POLQ and CHEK2, whereas only pathogenic phenotype are shown in Supplementary Table 1. and likely pathogenic variants were found in FANCM, CDKN2A and The potentially pathogenic VUS found in FANCM (c.5501G>A PTEN. Interestingly, potentially pathogenic VUS were commonly (p.Arg1834His)) and APC (c.3949G>C (p.Glu1317Gln)) were identi- found in FANCC, CFTR and RAD54L. CFTR has been previously associ- fied in 2 breast cancer cases from HBOC+PC families. The same APC ated with pancreatic cancer risk, whereas the association with FANCC variant was also found in a PDAC case from a different family. The and RAD54 variants is unclear. Patients with cystic fibrosis have an remaining cases with breast, liver, colon and thyroid tumours, ampu- increased lifetime risk of around 6 fold of developing tumours of the loma and pancreatitis were negative for pathogenic, likely pathogenic digestive tract, including pancreatic cancer. This is thought to be a or potentially pathogenic VUS. Potentially pathogenic VUS in MUTYH consequence of the damage caused to the pancreas by chronic pan- (c.1187G>A (p.Gly396Asp)), CFTR (c.3154T>G (p.Phe1052Val)) and creatitis and inflammation that commonly manifest in these patients the splice site variant in CHEK2 (c.592+50A>T (SPLICE SITE) were [27]. CFTR pathogenic variants are more common among PC cases identified in an individual diagnosed with a pancreatic neuroendo- compared to controls and a previous study has shown that common crine tumour from a family with multiple cases of colon cancer and CFTR mutations were detected in 5.3% of cases vs. 3.8% of controls also pancreatic cancer (Supplementary Table 1). [28]. There is a modest increased PC risk associated with CFTR muta- One high risk individual with an IPMN lesion identified during tions (odds ratios 1.4), particularly among smokers and those diag- secondary screening had a potentially pathogenic VUS splice site var- nosed at a younger age, with an odds ratio of 1.82 [28,29]. iant in ATM (c.7630 3C>T) and another high risk individual with a Germline mutations in the DNA repair genes such as BRCA, RAD51 pancreatic cyst had a potentially pathogenic VUS variant in BUB1 and POLQ are associated with hereditary breast cancer [30]. Interest- (c.341G>A (p.Arg114Gln). Two high risk individuals with no relevant ingly in this study, pathogenic BRCA2 mutations were generally found pancreatic lesions identified during follow-up had the potentially in the context of HBOC syndrome with at least of one case of PDAC pathogenic VUS variant in FANCM (c.1642G>C (p.Glu548Gln)) and and were not detected in any of the pure pancreatic cancer families. ATM (c.8560C>T (p.Arg2854Cys)), respectively. 2 high risk individu- The Fanconi anaemia (FA) pathway genes are related with DNA dam- als from the same FPC family (with 8 cases of PC) had 2 potentially age and repair and mono-allelic mutations in these genes increases pathogenic VUS variants in MSH6 (c.1857A>C (p.Glu619Asp)) and the risk of several types of cancer in a sporadic fashion. In fact, the CFTR ((c.2758G>T (p.Val920Leu)). Three individuals with pancreatic FANCD1 gene is actually BRCA2 [31]. FANCM and FANCC form part of tumours diagnosed during secondary screening were negative for the FA nuclear core protein complex, which consists of eight proteins, pathogenic, likely pathogenic or potentially pathogenic VUS (Supple- with ubiquitin ligase activity that is required for the monoubiquitina- mentary Tables 1 and 3). tion of FANCD2 in response to DNA damage. The FANCM likely patho- 3 potentially pathogenic VUS were found in an unaffected individual genic variant c.5791C>T (p.Arg1931Ter) found in one case in this with a pathogenic BRCA2 germline mutation (c.6959T>A (p.Leu2320Ter)) study is associated with non-BRCA1/BRCA2 related breast cancer and in ATM (c.998C>T (p.Ser333Phe)), BUB1 (c.341G>A (p.Arg114Gln)) and has also been recently described in an individual from a FPC family BRCA2 (c.8850G>T (p.Lys2950Asn)). The same BRCA2 potentially patho- who was diagnosed with breast cancer at age 45 and PC some genic VUS was also found in the sibling who also carried the same patho- 20 years later (32). It is also of interest that the FANCC potentially genic BRCA2 pathogenic variant and also had a cystic pancreatic lesion pathogenic VUS c.584A>T (p.Asp195Val) was also found in 4 cases. that was identified during screening (Supplementary Table 1). POLQ at 3q13.31 codes for, pol u , a translesional DNA polymerase According to the chi-square analysis, the frequency of potentially involved in double strand break repair [30]. Therefore, the p.P1514fs*3 pathogenic VUS variants was higher in PDAC versus non-PDAC cases, POLQ likely pathogenic variant found in this study is a strong causative which was statistically significant (chi-square statistic: 4.3797, gene candidate in the described case. The RAD genes play an important p = 0.036). There was no significant difference in potentially pathogenic role in homologous recombination and defects in these genes are associ- VUS variant frequency between PDAC cases and high risk individuals. ated with chromosomal instability and also lymphoma predisposition [33]. However, pathogenic variants in these genes have not yet been 4. Discussion associated with FPC. Therefore, it is of interest that the potentially path- ogenic VUS c.1487G>C (p.G496A) was identified in 2 PDAC cases and a This study shows that an important number (21%) of hereditary or breast cancer case from 3 different families considering that the fre- familial pancreatic cancer cases harbour pathogenic and likely patho- quency of this variant in the general population is 0.002 (https://www. genic variants in previously described genes associated with heredi- genome.gov/10001688/international-hapmap-project/). CHEK2 (Check- tary cancer. In this study, 7% of PDAC cases from FPC families had a point kinase 2) is a serine/threonine kinase that forms part of the DNA pathogenic mutation in the genes CDKN2A and MLH1 that are tradi- damage response pathway. Germline mutations in CHEK2 have been tionally associated with Lynch syndrome and hereditary melanoma. associated with prostate and breast cancer [34]. The likely pathogenic The CDKN2A variant (c.176T>G; p.Val59Gly) was identified in a case variant c.409C>T (p.Arg137Ter) found in this study is in the FHA from an FPC family with more than 3 cases of PC and was previously domain of CHEK2. The variant rs1801166 in APC (c.3949G>C (p. described in association with head and neck cancers. CDKN2A germ- Glu1317Gln)) is classified as conflicting interpretation of pathogenicity line mutations are rare in FPC families. However, members of familial in ClinVar, although it may have pathogenic potential and was found in atypical multiple mole melanoma (FAMMM) syndrome families are one PC case and one breast case from different families [35]. at a higher risk of developing PC and should therefore be offered Germline mutations in PTEN have been rarely described in PC. screening for early detection [14,23,24]. A predisposition to PC is Recently, exome sequencing identified the missense mutation, observed in individuals with Lynch syndrome (LS), which is charac- p.Arg234Gln in PTEN in a female diagnosed with a solid pancre- terised by pathogenic variants in the mismatch repair genes MLH1, atic tumour but without characteristics of PTEN hamartoma MSH2, MSH6 and PMS2. The pathogenic MLH1 variant c.1852A>G (p. tumour syndromes (PHTS). Loss of PTEN expression and overex- Lys618Glu) was found in 2 cases from 2 different families and the pression of TP53 was also confirmed by IHC in the primary potentially pathogenic VUS c.52A>G (p.Ile18Val) in PMS2 was found tumour and the somatic KRAS p.Gly12Val mutation was also iden- in 2 cases from 2 different families. LS individuals have an 8.6-fold tified, which is characteristic of a PDAC [36]. However, the 2 trun- increased risk of PC compared with the general population and upper cating variants found in the PDAC cases in this study have not gastrointestinal tract tumours predominantly occur in MLH1 patho- been previously described in familial cancer cases. TET proteins genic variant carriers [25,26]. (Ten-Eleven Translocation proteins) are enzymes involved in DNA J. Earl et al. / EBioMedicine 53 (2020) 102675 7 demethylation and inactivation can lead to promoter hyperme- the risk of pancreatic cancer development. However, their role in pan- thylation and somatic and germline alterations in these genes creatic cancer risk needs to be clarified in other cohorts. have been implicated in myeloid and lymphoid tumours [37]. Interestingly, 2 likely pathogenic variants were found in this gene 5. Conclusion in one case from a HBOC+ PC family. The potentially pathogenic VUS c.1187G>A (p.Gly396Asp) in MUTYH was found in 2 cases from 2 different families, one pure FPC family and  Pathogenic and likely pathogenic variants were identified in 19% one family with HBOC with PC. Pathogenic variants have been previ- of pure FPC families (MLH1, CDKN2A, POLQ, TET2, FANCM). Poten- ously described in this gene in PC cases, although not in association tially pathogenic VUS were identified in 31% of pure FPC families with FPC [16]. In this study, we also identified high risk individuals with (FANCC, MLH1, PMS2, CFTR, MUTYH) potentially pathogenic VUS in this gene. BUB1 forms part of the compo-  The genes associated with familial PDAC in this study included nents of the spindle assembly checkpoint (SAC) and controls chromo- CDKN2A, MLH1, BRCA2, POLQ, CHEK2, PTEN, TET2, FANCM, FANCC, some segregation and germline mutations have been reported in RAD54L, PMS2 and MUTYH. hereditary colorectal cancer (CRC). BUB1 germline mutations have been  These results could contribute to the design of a potential useful previously described in pancreatic cancer cases, although the potentially genetic signature for screening programs in a high-risk population. pathogenic VUS found in a high risk individual in this study has not pre- viously described in familial cancer [38,39]. Declaration of Competing Interest The majority of germline truncating variants found in this study have not been described previously in the context of pancreatic can- None. cer [14,16,17,18,40,41]. Around 20% of cases included in this study had more than one pathogenic, likely pathogenic or potentially path- Acknowledgments ogenic VUS in the genes studied. The presence of more than one dele- terious variant also been previously described in FPC [42]. The genes We would like to thank the collaboration of the clinical and associated with familial PDAC in this study included CDKN2A, MLH1, nursing staff that have contributed to this project, Carmen Guillen BRCA2, POLQ, CHEK2, PTEN, TET2, FANCM, FANCC, RAD54L, PMS2 and Ponce, María Teresa Salazar Lo pez, Andrea Santos Gil, Maria del MUTYH, which is consistent with previous reports of panel sequenc- Carmen Perez Ruiz, Manuela Hernando, Maria Jesus Casado Ces- ing in these individuals [14]. pedosa and Clara Pacheco Torres. Furthermore, we would also With regard to the distribution of pathogenic, likely pathogenic like to thanks Francisco Del Castillo for help with the sequencing and potentially pathogenic VUS in different cancer syndromes, our analysis and Alfonso Muriel for the statistical analysis. data show that some genes are implicated in both FPC and HBOC syn- This study was funded by the Instituto de Salud Carlos III drome cases, whereas, other genes are associated with either FPC or (Plan Estatal de I+D+i 2013-2016): ISCIII (PI09/02221, PI12/01635, HBOC cases. POLQ, FANCM, CDKN2A and PTEN pathogenic and likely PI15/02101 and PI18/1034) and co-financed by the European Devel- pathogenic variants were only found in FPC cases, whereas TET2 and opment Regional Fund ‘‘A way to achieve Europe’’ (ERDF), the Bio- CHEK2 variants were only found in HBOC cases. However, pathogenic, medical Research Network in Cancer: CIBERONC (CB16/12/00446), likely pathogenic and potentially pathogenic VUS in MLH1 were Red Tema tica de investigacio  n cooperativa en cancer: RTICC (RD12/ found in both FPC and HBOC cases. With regard to potentially patho- 0036/0073) and La Asociacio n Espan~ ola contra el Cancer: AECC (Gru- genic VUS, these were more frequently found in the genes MUTYH, pos Coordinados Estables 2016). TET2 and CHEK2 in HBOC cases, whereas potentially pathogenic VUS The funding sources of this study did play any role in the study design, were most frequently found in PMS2, POLN and MSH2 in FPC cases. the collection, analysis and interpretation of the data, in the writing of the There are some limitations of the study. Firstly, only 43 PDAC familial manuscript and in the decision to submit the paper for publication. cases were included due to the rare nature of this syndrome. Therefore The corresponding author has full access to all the data in the study it would be ideal to validate these findings in an independent cohort and assumes the responsibility for the decision to submit for publication. from another international registry. Furthermore, it would be interest- ing to perform the same study in sporadic PDAC cases as there is Supplementary materials increasing evidence that these cases may also harbour germline risk var- iants. In fact, an estimated 4% 10% of patients with pancreatic cancer Supplementary material associated with this article can be found harbour mutations in hereditary pancreatic cancer susceptibility genes in the online version at doi:10.1016/j.ebiom.2020.102675. [43]. Although, due to the frequency of sporadic cases with germline mutations, it is possible that these are actually familial cases that have References not been adequately identified as such. 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