inactivation of the mismatch repair ( mmr ) genes mlh1 , msh2 , msh6 and pms2 causes lynch syndrome ( ls ) , an autosomal dominant predisposition for colorectal and endometrial cancer .
inactivation of the mismatch repair pathway can also occur sporadically , through somatic mlh1 hypermethylation or by acquired biallelic somatic inactivation ( variant affecting function or loss of heterozygosity ( loh ) ) of the mmr genes .
inaccurate dna repair leads to a high frequency of somatic variants , with loss of mmr leading to  hypermutated ' tumors with 10100 variants / mb .
ls tumors are characterized by microsatellite instability ( msi ) and immunohistochemical loss of expression of mmr proteins .
however , germline variants affecting function can not be detected in up to 59% of patients displaying msi and/or loss of mmr , referred to as  suspected ls ' ( sls ) .
recently , germline and somatic variants in the exonuclease domains ( edms ) of dna polymerase  ( pole ) and polymerase d ( pold1 ) were described .
these pole / pold1 variants affect proofreading function and lead to an ultramutated phenotype with a variant incidence exceeding 100 variants / mb .
germline pole - edm variants can result in a ls phenotype and microsatellite instable colorectal cancers ( crcs ) .
the exact role of somatic pole / pold1 variants in tumors with high microsatellite instability ( msi - h ) remains unclear .
the aim of our study was to identify the underlying genetic cause of disease in a cohort of 64 sls cases  selected on the basis of msi , loss of mmr , young onset and often a family history for ls  by screening the mmr , pole and pold1 genes in both leukocyte and tumor dna .
this study included 64 patients with lynch - associated tumors recruited in four academic centers in the netherlands between 1997 and 2014 : leiden university medical centre ( n=37 ) , maastricht university medical centre ( n=11 ) , erasmus medical centre ( n=9 ) and university medical centre utrecht ( n=7 ) .
demographic and clinical data , as well as informed consent , were obtained at the time of diagnosis .
this study was approved by the local medical ethical committee of the lumc ( p01 - 019e ) .
patients were selected based on loss of mmr ( as indicated by immunohistochemical staining ) and/or msi .
unexplained tumors with low msi or tumors with inconclusive ihc results were also included in this study ( see supplementary table 1 and supplementary methods ) .
fifty - eight ( 91% ) patients fulfilled bethesda criteria , and families of 24 ( 38% ) patients also fulfilled amsterdam ii criteria .
patients were previously screened in a diagnostic setting for germline mmr variants . whereas 57 patients showed no disease - causing germline variants , 7 patients were found to have a germline variant of unknown significance ( vus ) . of the total cohort , 75% of patients presented with crc ( n=48 ) , 14% with endometrial cancer ( ec , n=9 ) and 11% with another ls - associated tumor
two patients were excluded from the analysis because of poor dna quality . of the remaining 62 tumors , tumor and
leukocyte dna was sequenced for variants in the exonic regions of mlh1 , msh2 , msh6 , pms2 , pole and pold1 using the ion pgm system ( life technologies , carlsbad , ca , usa ) .
raw data analysis , alignments and variant calling was carried out using the default parameters in torrent suite v4.0 ( thermo fisher scientific , waltham , ma , usa ; see supplementary methods ) .
the full data set was filtered and prioritized by variant frequency ( > 10% ) and coverage ( > 50  ) .
the in silico prediction programs were used to predict effect on function ( see supplementary methods ) .
all variants ( likely ) affecting function , including two variants with a 9% variant frequency , were validated with sanger sequencing . for all pms2 variants ,
pms2-specific primers were created to confirm that the variant is present in pms2 and not in a pms2 pseudogene .
loh was determined for every heterozygous snp by comparing the ratio of allele a with allele b in leukocyte and tumor dna samples .
furthermore , for every heterozygous snp , the allelic imbalance factor ( aif ) was calculated and fisher 's exact test was performed to determine whether the difference between normal and tumor is significant . if all heterozygous snps of one gene showed loh with an aif > 2 and fisher 's exact p - value < 0.05 , loh was called ( supplementary table 1 ) .
seven patients with a germline mmr vus ( class 3 ) were included in this study ( see supplementary table 1 ) . in all cases ,
the variant was detected with ngs in leukocyte and tumor dna . during the course of the study ,
three of these germline variants were reclassified as class 4 or 5 ( ( probably ) affects function ) by the international society for gastrointestinal hereditary tumors incorporated ( insight ) .
six cases with somatic mlh1 hypermethylation fulfilling revised bethesda criteria ( three from families fulfilling amsterdam ii criteria ) were sequenced for underlying hereditary defects explaining the family history , but no germline variants were found .
one of these mlh1 hypermethylated tumors carried a somatic mlh1 variant likely to affect function and one displayed mlh1 loh ( supplementary table 1 ) .
one ( n=27 , 44% ) or two ( n=13 , 21% ) somatic aberrations ( variant or loh ) in a mmr gene were found in a total of 62 tumors ( see supplementary table 1 ) .
of the 13 tumors with two somatic aberrations , 12 had variants in mlh1 or msh2 and were msi - h .
although the majority ( 83% ) of tumors showed < 10 somatic variants in the genomic region analyzed , 10 cases displayed a larger number of somatic variants , ranging from 16 to 375 somatic variants within the sequenced area of 31  kb .
out of 10 tumors , 9 showed a pole or pold1 variant that ( probably ) affects function ( table 1 ) . of the highly mutated tumors , two carried novel germline heterozygous pole / pold1 variants that are predicted to affect proofreading ( supplementary table 2 ) .
of these two germline cases , tumor sls-67 was also found to carry two somatic mlh1 variants , explaining the tumor phenotype ( loss of mlh1 and pms2 expression and msi - h ) .
the second tumor , sls-16 , was msi - l , showed positive mlh1 and msh2 staining ( msh6 and pms2 were not tested ) and had no somatic mmr variants .
seven of the highly mutated tumors showed somatic pole / pold1 variants likely to affect function .
six tumors carried a somatic pole / pold1-edm hot spot variant ( pole : c.857c > g , c.856c > t , c.1231g > t , c.1366g > c , c.1367c > t or c.1376c > t and pold1 c.1433g > a ) that has previously been described to impair proofreading . in the seventh tumor ( sls-105 ) , a novel pole c.846_847delinstt variant was detected .
this variant lies close to a known pole hot spot site ( pole c.857 ) and is predicted to affect function by two out of three prediction programs ( supplementary table 2 ) .
four pole / pold1-edm mutated tumors displayed additional somatic nonsense pole variants outside the exonuclease domain ( see supplementary table 3 ) .
eight of the nine ultramutated tumors with a pole / pold1-edm variant found in our study showed msi ( 3 msi - h and 5 msi - l ) . in six of these ultramutated tumors ,
ihc detected loss of at least one of the mmr proteins and all six tumors displayed somatic variants in the affected mmr gene likely to affect function .
tumor sls-19 with two pole variants was found to have two somatic mlh1 aberrations , as well as two somatic pms2 aberrations , and ihc showed solitary loss of pms2 expression .
reanalysis of staining also showed ambiguous mlh1 staining ( cytoplasmic enhancement and vague , focal nuclear staining ) .
tumor sls-09 displayed a nonsense msh6 variant and missense msh2 variant predicted to affect function , and ihc showed loss of msh6 expression and weak positive msh2 expression . in three pole
/ pold1-edm mutated tumors with positive or inconclusive mmr expression and msi - l phenotype , no somatic mmr variants ( likely ) to affect function were found .
however , in one of those three tumors ( sls-05 ) , solitary mlh1 loh and pms2 loh without variants was found ( see table 1 ) . in three non - ultramutated tumors ,
a pole / pold1-edm variant was found ( sls-80 , sls-87 and sls-101 , see table 1 ) .
these variants have not been described before , but are predicted to affect function ( see supplementary table 2 ) .
two variants co - occur with a germline msh6 vus and a somatic msh6 variant ( sls-80 and sls-87 ) , whereas one ( sls-101 ) co - occurs with a somatic pms2 variant .
four additional non - ultramutated tumors showed pole ( sls-18 , sls-21 ) or pold1 loh ( sls-12 , sls-49 ) in all heterozygous snps ( supplementary table 1 ) , without germline or somatic pole variants .
pole - edm variants are reported to be the mutagenic factor driving ultramutation in tumors .
the same report also noted that the increased mutational load seen in pole mutated tumors exceeds that expected because of loss of exonuclease activity .
the number of variants detected in the sequenced area in the present study implicates an ultramutated phenotype , with > 100 variants / mb in all pole / pold1-edm mutated tumors in this cohort . as only a limited region ( 31  kb )
was sequenced , we can only extrapolate the total number of variants per mb . in our cohort ,
the pole / pold1 mutated mmr - deficient tumors display two deficient pathways increasing the mutational load . comparing frequencies of the different variants found in these tumors ,
it might be concluded that faulty proofreading may be the initiating event in some of these tumors , possibly resulting in loss of mmr and thereby in msi .
interestingly , four tumors show pole / pold1 loh without germline or somatic pole / pold1 variants .
these tumors however do not show the typical ultramutated phenotype , whereas single variants without loh do show that .
this phenomenon of loh without variants affecting the exonuclease domain has not yet been described .
furthermore , three tumors show somatic pole / pold1 variants , without the ultramutated phenotype .
all three variants are missense , but are predicted to affect function ( supplementary table 2 ) . as these variants are not found in ultramutated tumors , evidence of deleterious functional effect is lacking . in conclusion , targeted next - generation sequencing of 62 sls cases led to the detection of 9 highly mutated tumors with a germline ( n=2 ) or somatic ( n=7 ) pole / pold1-edm variant . even though pole germline variants have previously been shown to co - occur with somatic mmr variants , in this study we found germline and somatic pole / pold1 variants in a cohort selected for sls characteristics .
importantly , although current literature mainly addresses pole / pold1 variants in mss tumors , somatic pole / pold1 variants in sls patients are likely to be overlooked . however , a very recent recommendation for genetic testing and surveillance states that mmr deficiency should not be an exclusion criterion for genetic testing of germline pole / pold1 variants .
our results further emphasize the importance of pole / pold1 germline and somatic screening in unexplained msi - h and mmr - deficient tumors .
 many suspected lynch syndrome ( sls ) patients who lack mismatch repair ( mmr ) germline gene variants and mlh1 or msh2 hypermethylation are currently explained by somatic mmr gene variants or , occasionally , by germline pole variants . to further investigate unexplained sls patients , we analyzed leukocyte and tumor dna of 62 sls patients using gene panel sequencing including the pole , pold1 and mmr genes . 
 forty tumors showed either one , two or more somatic mmr variants predicted to affect function . 
 nine sls tumors showed a likely ultramutated phenotype and were found to carry germline ( n=2 ) or somatic variants ( n=7 ) in the pole / pold1 exonuclease domain ( edm ) . 
 six of these pole / pold1-edm mutated tumors also carried somatic mmr variants . 
 our findings suggest that faulty proofreading may result in loss of mmr and thereby in microsatellite instability . 