Introduction

Duodenal, ampullary, and pancreatic carcinomas (DC, AC, and PC, respectively) are rare gastrointestinal (GI) malignancies, typically associated with late age of onset [1]. A minority develops in young patients [1], partly within Lynch syndrome (LS) [2]. LS, previously referred to as hereditary non-polyposis colorectal cancer (HNPCC), is a major hereditary cancer syndrome with an autosomal dominant pattern of inheritance, caused by heterozygous pathogenic germline variants in DNA mismatch repair (MMR) genes (MLH1, MSH2/EPCAM, MSH6, and PMS2) [2]. LS-associated malignancies arise after the subsequent somatic inactivation of the remaining wild-type allele of the affected MMR gene leading to impaired DNA MMR and accumulation of replication errors. Deficient DNA MMR (dMMR) is characterised by absence of MMR proteins and leads to microsatellite instability (MSI), a molecular phenotype characterised by accumulation of multiple alterations within microsatellite repeat regions throughout the genome [2]. The presence of MSI/dMMR in tumours can be based on germline, as in LS or constitutional mismatch repair deficiency (CMMRD) syndrome, or somatic MMR gene defects, such as silencing of MLH1 by promoter hypermethylation or due to biallelic pathogenic somatic variants in MMR genes, resulting in loss of corresponding MMR protein expression [2]. LS patients have an increased lifetime risk to develop colorectal cancer (CRC) and a variety of extracolonic malignancies [2]. Since early age of cancer onset can be a hallmark of an underlying hereditary condition [3], in this study, we investigated the incidence of dMMR in patients diagnosed with DC, AC, and PC before the age of 50 years (<50).

Materials and methods Patient cohorts

We performed a nationwide retrospective search (LZV977) in the Nationwide Network and Registry of Histopathology and Cytopathology in the Netherlands (PALGA) [4], with approval of their Privacy Commission and Scientific Council, to identify all patients diagnosed with primary DC and PC under the age of 50 in the Netherlands between January 2002 and December 2012. To have sufficient tissue for analyses, only resection specimens were requested. A substantial number of patients initially diagnosed with PC had, in fact, AC. Considering their high prevalence in our cohort (n = 23), and because ACs have generally better prognosis compared to conventional pancreatic adenocarcinomas [5], they were categorised separately. Normal and tumour tissue materials (formalin-fixed paraffin-embedded [FFPE] tissue blocks) were requested from eligible cases, resulting in 23 cooperating laboratories throughout the Netherlands. Additional information including the nationwide personal pathology history was requested for all included patients (contains information up to February 2020).

Findings in early-onset DC patients were compared to the internal Radboud University Medical Center cohort of late-onset (diagnosed above the age of 50 years) DC patients (DC ≥ 50) (n = 18).

Tissue materials were reviewed by pathologist MAJM-vZ, and clinicopathological characteristics were extracted from individual pathology reports.

This study (CMO-2017-3780) was approved by the local ethical committee of the Radboud University Medical Center. Personal data concerning individual patients were anonymised prior to obtaining patient data and tissue materials, thereby preventing identification of the individuals included in the study. In consequence, patient consent was not required, and the results of germline analyses could not be shared with patients, their families, and physicians.

Research strategy

Immunohistochemical staining for MLH1, MSH2, MSH6, and PMS2 was used to determine dMMR status. In cases with aberrant staining, MSI analysis was performed, followed by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) to detect somatic MLH1 promoter hypermethylation in cases with MLH1 and/or PMS2 loss. Single-molecule molecular inversion probes (smMIP) sequencing for the MMR genes was performed on normal and tumour DNA. MLPA analysis to detect exon deletions and duplications was performed on cases without pathogenic variants or with only one somatic event [6].

IHC, MSI analysis, and somatic hypermutation

Immunohistochemistry (IHC) for MMR (MLH1, MSH2, MSH6, and PMS2) protein expression was performed using standard procedures. Tissue microarray slides were stained with antibodies against MLH1 (clone G168-15; BD Biosciences, San Jose, CA, USA), MSH2 (clone GB12; Calbiochem/Merck, Darmstadt, Germany), MSH6 (clone EPR3945; Abcam, Cambridge, UK), and PMS2 (clone A16-4; BD Biosciences, San Jose, CA, USA). Based on the IHC pattern, all tumours were classified as MLH1-deficient (aberrant MLH1 and PMS2 staining), PMS2-deficient (loss of PMS2 staining), MSH2-deficient (aberrant MSH2 and MSH6 staining), and MSH6-deficient (loss of MSH6 staining). Scoring was performed by two blinded observers (MAJM-vZ and IDN) as described by Overbeek et al [7].

Genomic DNA was extracted from deparaffinised FFPE normal and tumour tissue for all cases with aberrant expression of at least one MMR protein, according to standard procedures. Regions with at least 30% neoplastic cells (when possible) were dissected for tumour DNA, as required for sensitive MSI detection. MSI was assessed using five mononucleotide markers BAT25, BAT26, NR21, NR24, and NR27 as described previously [8]. Tumours without any unstable markers were categorised as microsatellite stable (MSS), and cases with more than one unstable marker as having a high degree of MSI (MSI-H). In cases with doubtful findings or low tumour cell percentage, MSI assessment was performed using targeted smMIP-based next-generation sequencing (NGS) PATHv2D panel including 55 sensitive markers for MSI detection. Library preparation and sequencing using smMIP-based libraries were performed on a NextSeq 500 (Illumina, San Diego, CA, USA) according to the manufacturer's instructions as described previously [9]. The presence of ≥30% of unstable markers out of the total assessable was scored as MSI.

To detect somatic MLH1 promoter hypermethylation, MS-MLPA was performed on tumour DNA from MLH1- and PMS2-deficient cases using standard procedures during routine diagnostic procedures (MRC-Holland, Amsterdam, The Netherlands).

Germline and somatic mutation analyses Targeted sequencing

Targeted smMIP-based NGS was performed on normal and tumour DNA. Library preparation and sequencing using NextSeq 500 approach were performed as described previously [9]. The entire coding regions of four MMR genes, MLH1 (NM_000249.3), MSH2 (NM_000251.2), MSH6 (NM_000179.2), and PMS2 (NM_000535.5) were sequenced. MLH1- and PMS2-deficient cases were initially sequenced for MLH1, followed by PMS2 if no MLH1 variants were detected. All MSH2- and MSH6-deficient cases were sequenced for MSH2 and MSH6. Sequencing reads were aligned to the reference genome (human genome 19). Variants were called and sequencing results were analysed using Sequence Pilot (JSI Medical Systems, Ettenheim, Germany) software for genetic analysis as described previously [9]. Identified variants were evaluated with Alamut Visual version 2.13 (SOPHiA GENETICS, Lausanne, Switzerland) software and publicly available databases such as ClinVar [10] and InSiGHT [11], and categorised based on the current guidelines for variant classification defined by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology [12].

MLPA assays

All MSI/dMMR cases without detectable pathogenic variant or with one identified somatic event underwent MLPA analysis to detect exon deletions and duplications depending on the affected MMR gene based on NGS. MLPA was performed on normal and tumour DNA using the MRC-Holland SALSA probe mix assays (MRC-Holland) according to the manufacturer's instructions.

Diagnostic criteria

Based on germline and somatic mutation analyses, all cases were classified as having: (1) CMMRD syndrome when there were biallelic pathogenic germline variants in one of the MMR genes. Aberrant IHC expression of the affected protein in normal tissue confirmed the presence of biallelic hits. The personal history of patients was studied to identify other malignancies concordant with CMMRD to support the diagnosis; (2) LS when a pathogenic germline variant was detected in normal DNA, confirmed (when possible) by a somatic inactivating event (second somatic pathogenic variant or loss of heterozygosity [LOH]) in tumour DNA; (3) Unclassified when there was a pathogenic somatic event in tumour DNA but germline DNA could not be assessed due to the limitations of material; (4) Non-hereditary or sporadic when the causative pathogenic variant was present in tumour, but not in normal DNA, together with the second inactivating event, by means of second somatic hit or LOH.

Statistical analysis

Demographics, clinical data, and pathological characteristics were analysed. Chi-square (χ2) test or Fisher's exact test (if observed or expected sample size in the contingency table was less than 5) was used for categorical data; the Kruskal–Wallis test was used for continuous variables. Two-sided P values of <0.05 were considered as statistically significant. Statistical analyses were calculated only using patients with sufficient available data. All analyses were performed using the SPSS software (IBM SPSS Statistics, version 25 [SPSS Inc., Chicago, IL, USA]).

Results

Of the 162 identified patients, 90 were included (Figure 1A), and their characteristics are summarised in Table 1. Tumours diagnosed before the age of 30 years were only present in the DC group (supplementary material, Table S1).

(A) Patient selection and (B–E) molecular background of dMMR cases. Almost half of early-onset DCs exhibited MSI/dMMR, which was strongly associated with underlying hereditary MMR defect due to either CMMRD or LS (B). MSI/dMMR was rare in young AC (C) and PC (D) patients but, when present, strongly indicative of LS. In contrast to early-onset, late-onset DCs rarely showed MSI/dMMR, and a single dMMR case was non-hereditary (E).

Table 1. Clinicopathological characteristics and MSI status of the patients. Early-onset cases (<50 years) P values Late-onset cases (≥50 years) All, n = 90 Duodenal, n = 23 Ampullary, n = 23 Pancreatic, n = 44 DC versus AC AC versus PC DC versus PC Duodenal, n = 18 P value Gender, n (%) Male 53 (58.9) 15 (65.2) 10 (43.5) 28 (63.6) 0.139 0.114 0.898 13 (72.2) 0.632 Female 37 (41.1) 8 (34.8) 13 (56.5) 16 (36.4) 5 (27.8) Age, median (range) (years) 43 (17–49) 46 (17–49) 44 (33–49) 43 (30–49) 0.691 0.005 0.174 70 (57–77) <0.001 Histological type, n (%) Adenocarcinoma NOS 84 (93.3) 20 (87) 21 (91.3) 43 (97.7) >0.999 0.114 0.113 14 (77.8) 0.654 Mucinous 3 (3.3) 1 (3.4) 2 (8.7)* 0 2 (11.1) Signet ring cell 1 (1.1) 1 (3.4) 0 0 2 (11.1) Adenosquamous 1 (1.1) 0 0 1 (2.3) 0 Medullary 1 (1.1) 1 (3.4) 0 0 0 Diameter, median (range) (cm)† 3 (0.5–7) 4.4 (0.9–6) 2 (0.5–7) 3 (1.5–6.5) 0.004 0.01 0.028 4 (1.4–13) 0.946 Differentiation grade, n (%) Well/moderate 57 (63.3) 14 (60.9) 13 (56.5) 30 (68.2) 0.765 0.345 0.549 12 (66.7) 0.702 Poor 33 (36.7) 9 (39.1) 10 (43.5) 14 (31.8) 6 (33.3) T-stage, n (%)† T1 5 (5.6) 2 (8.7) 3 (13) 0 0.018 <0.001 <0.001 1 (5.6) 0.304 T2 10 (11.1) 0 6 (26.1) 4 (9.1) 3 (16.7) T3 58 (64.4) 10 (43.5) 9 (39.1) 39 (88.6) 7 (38.9) T4 15 (16.7) 11 (47.8) 4 (17.4) 0 7 (38.9) Unknown 2 (2.2) 0 1 (4.3) 1 (2.3) 0 N-stage, n (%)† N0 28 (31.1) 11 (47.8) 7 (30.4) 10 (22.7) 0.014 0.328 <0.001 9 (50) 0.065 N1 52 (57.8) 6 (26.1) 13 (56.5) 33 (75) 9 (50) N2 5 (5.6) 5 (21.7) 0 0 0 Unknown 5 (5.6) 1 (4.3) 3 (13) 1 (2.3) 0 Overall MSI/dMMR, n (%) MSS/pMMR 76 (84.4) 12 (52.2) 22 (95.7) 42 (95.5) 0.002 >0.999 <0.001 17 (94.4) 0.005 MSI/dMMR 14 (15.6) 11 (47.8) 1 (4.3) 2 (4.5) 1 (5.6) Immunohistochemical analysis of MSI/dMMR cases, n (% MSI/dMMR) MLH1/PMS2 4 (28.6) 3 (27.3) 1 (100) 0 0.5 0.333 0.026 1 (100) 0.5 PMS2 6 (42.9) 6 (54.5) 0 0 0 MSH2/MSH6 2 (14.3) 2 (18.2) 0 0 0 MSH6 2 (14.3) 0 0 2 (100) 0 Molecular background of MSI/dMMR cases, n (% MSI/dMMR) CMMRD 2 (14.3) 2 (18.2) 0 0 >0.999 NA >0.999 0 0.583 LS 8 (57.1) 5 (45.5) 1 (100) 2 (100) 0 Unclassified 2 (14.3) 2 (18.2) 0 0 0 Non-hereditary 2 (14.3) 2 (18.2) 0 0 1 (100) Fisher's exact test was used when at least one expected or observed value was below 5; in other cases, chi-square (χ2) test was used. Values in bold indicate statistically significant results (significance considered at p < 0.05). NA, not applicable; NOS, not otherwise specified. * Both mucinous and partially signet ring cell ACs. † Calculated only using patients with sufficient data available for certain characteristics.

Almost half of DC < 50 (11/23) exhibited dMMR (Figure 1B, Table 2). Two patients (2/11) had CMMRD syndrome caused by biallelic pathogenic germline variants in MMR genes, five patients (5/11) had LS, two (2/11) were categorised as unclassified due to insufficient quality of normal tissue for germline testing, and two (2/11) had biallelic somatic MMR aberrations (Table 2). Both CMMRD patients had a personal history of associated malignancies (Table 2). Only 1 of 18 DC ≥ 50 was dMMR due to biallelic somatic inactivation of MLH1 (Figure 1E, Table 2).

Table 2. Molecular background of MSI/dMMR DCs, ACs, and PCs. Patient ID Histological type and differentiation Sex Age Gene Germline Somatic Diagnosis Other tumours (age) DC < 50 DC11 Adenocarcinoma NOS, intestinal M 17 PMS2

c.137G>T p.(Ser46Ile) (class 4, likely pathogenic)

and

c.2174+1G>A p.? (splice site) (class 5, pathogenic)

None CMMRD Serrated adenoma colon (15), 3× tubulovillous adenoma duodenum (16, 17, 17), jejunal adenocarcinoma (19), 8× colon adenomas (20), tubulovillous adenoma jejunum (20), T-cell acute lymphoblastic leukaemia (21), multiple adenomas colon (22) DC12 Adenocarcinoma NOS, intestinal M 32 PMS2

c.(163+1_164-1)_(803+1_804-1) (exons 3–7 deletion) (class 5, pathogenic)

and

c.-87_(2174+1_2175-1) (exons 1–12 deletion) (class 5, pathogenic)

None CMMRD Colorectal adenomas (10, 10, 12, 15, 15, 16, 16, 17, 19, 21), CRC (21), adenomatous polyposis colon (21), duodenal adenoma (32), jejunal adenocarcinoma (32), ileal adenoma (32), diffuse astrocytoma with progression towards secondary glioblastoma (34) DC16 Adenocarcinoma NOS, intestinal M 46 PMS2 c.247_250dup p.(Thr84Ilefs*9) (class 5, pathogenic)

LOH and

c.1639dup p.(Ser547Phefs*15) (class 5, pathogenic)

LS Tubular adenoma rectum (60) DC13 Adenocarcinoma NOS, intestinal F 46 MSH2 c.1139delT p.(Leu380Tyrfs*32) (class 5, pathogenic) c.2332dup p.(Cys778Leufs*9) (class 5, pathogenic) LS CRC (24), CRC (26), endometrioid ovarian carcinoma (39), tubulovillous adenoma rectum (56), 3× tubular adenomas colon (58) DC10 Adenocarcinoma NOS, intestinal M 48 PMS2 c.736_741delinsTGTGTGTGAAG p.(Pro246Cysfs*3) (class 5, pathogenic) c.859dup p.(Arg287Lysfs*12) (class 5, pathogenic) LS Hyperplastic polyp sigmoid (52), hyperplastic polyp sigmoid and rectum (54), 2× tubular adenomas colon ascendens (57), prostate adenocarcinoma (58) DC17 Adenocarcinoma NOS, intestinal F 48 PMS2 c.(23+1_24-1)_(163+1_164-1)del (exon 2 deletion) (class 5, pathogenic) c.1A>G p.Met1? (class 4, likely pathogenic) LS Adenocarcinoma NOS breast (52) DC19 Adenocarcinoma NOS, intestinal F 49 MLH1 c.1896G>A p.? (splice site) (class 5, pathogenic) NA* LS Endometrial adenocarcinoma (55) DC14 Adenocarcinoma NOS, intestinal M 29 MSH2 NA† c.679_687delinsTTCCTAAAAA p.(Arg227Phefs*5) (class 5, pathogenic) Unclassified None DC23 Adenocarcinoma NOS, intestinal M 49 MLH1 NA† c.531_532delinsCT p.(Leu177_Glu178delinsPhe*) (class 5, pathogenic) Unclassified CRC (49), colon adenomas (49, 50, 53, 57, 57), hyperplastic polyp sigmoid (57) DC25 Adenocarcinoma, medullary M 34 PMS2 None c.338C>A p.(Ser113*), biallelic (class 5, pathogenic) Sporadic DC Tubular adenoma colon (34) DC1 Adenocarcinoma NOS, intestinal M 46 MLH1 None c.-198_*193del (entire gene deletion), biallelic (class 5, pathogenic) Sporadic DC Seminoma testis (24), colon adenomas (51, 52, 52, 52, 55, 55, 57, 57, 59, 59), 9× colorectal adenomas (62) DC ≥ 50 DC24c Adenocarcinoma NOS, intestinal M 66 MLH1 None c.2074_2078del p.(Ser692Glyfs*10) (class 5, pathogenic) and LOH Sporadic DC NI AC < 50 AC22