detecting genetic hypermutability of gastrointestinal

Detecting genetic hypermutability of gastrointestinal tumor byusing a forensic STR kit

Anqi Chen1,2,*, Suhua Zhang2,*, Jixi Li1, Chaoneng Ji1, Jinzhong Chen (✉)1, Chengtao Li (✉)2

1State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China;2Shanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Institute of Forensic Sciences, Ministry of Justice,Shanghai 200063, China

© Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract Growing evidence suggests that somatic hypermutational status and programmed cell death-1overexpression are potential predictive biomarkers indicating treatment benefits from immunotherapy usingimmune checkpoint inhibitors. However, biomarker-matched trials are still limited, and many of the genomicalterations remain difficult to target. To isolate the potential somatic hypermutational tumor from microsatelliteinstability low/microsatellite stability (MSI-L/MSS) cases, we employed two commercial kits to determineMSI andforensic short tandem repeat (STR) alternations in 250 gastrointestinal (GI) tumors. Three types of forensic STRalternations, namely, allelic loss, Aadd, and Anew, were identified. 62.4% (156/250) of the patients with GIexhibited STR alternation, including 100% (15/15) and 60% (141/235) of the microsatellite high instability andMSI-L/MSS cases, respectively. 30% (75/250) of the patients exhibited STR instability with more than 26.32%(26.32%–84.21%) STR alternation. The cutoff with 26.32% of the STR alternations covered all 15 MSI cases andsuggested that it might be a potential threshold. Given the similar mechanism of the mutations of MSI and forensicSTR, the widely used forensic identifier STR kit might provide potential usage for identifying hypermutationalstatus in GI cancers.

Keywords mismatch repair protein deficiency (MMR-D); microsatellite instability (MSI); short tandem repeats (STR);gastrointestinal tumor; hypermutability

Introduction

Mismatch repair protein deficiency (MMR-D) causes DNArepeat region hypermutation and instability [1]. Multipletests are available to evaluate MMR-D status [2]. Thesemethods include immunohistochemistry staining for mis-match repair proteins, MLH1 promoter methylationanalysis, and microsatellite instability (MSI) evaluation[3–6]. These tests exhibit prognostic and therapeuticimplications for patients with colorectal carcinoma andgastric and endometrial cancers [7]. In MSI, cancerproduces frame-shift mutations, yielding abnormal pro-teins and novel epitopes [8]. The accumulation of

mutations causes the development of high antigenicityimmune phenotype that may respond to immune check-point inhibitors. Approximately 5%–21% of colorectalcancers (CRCs) or/and gastric cancers are characterized bydifferent MSI methods [1,9,10]. Most MSI high (MSI-H)and/or PD-L1 positive cases respond to immune check-point inhibitors, whereas some MSI low/microsatellitestability (MSI-L/MSS) cases still respond to the agent [11–13]. This phenomenon suggests the existence of alternativeMSI forms (A-MSI) [14,15].Future tumor research with immune therapeutic

approaches will possibly focus on identifying strategiesto improve non-MSI-H tumor immunogenicity. Elevatedhypermutation alterations with improved MSI detectionmethods, such as elevated microsatellite alterations atselected tetranucleotide repeats (EMAST) and next-gen-eration sequencing (NGS)-based tumor mutation burden(TMB), can identify some hypermutation cases from theMSI-L group [16–18]. These methods may improve thebiomarker-matched trials and increase the number of

Received October 17, 2018; accepted April 13, 2019

Correspondence: Jinzhong Chen, [email protected];

Chengtao Li, [email protected]*The authors contributed equally to the work.

RESEARCH ARTICLEFront. Med. 2020, 14(1): 101–111https://doi.org/10.1007/s11684-019-0698-4

patients that benefit from immunotherapy with immunecheckpoint inhibitors. However, EMAST application waslimited by the lack of a consensus criterion, whereas theTMB measured by NGS was time- and cost-defective.Therefore, generating a rapid and convenient method foridentifying additional A-MSI in patients with tumor isurgently needed.Herein, we reported a study combined with two

commercial kits to determine the hypermutation status innumerous patients with gastrointestinal (GI) cancer toprovide a new potential biomarker to detect patients withgenetic hypermutability in addition to assess the MSIanalysis. This process may help screen patients who canbenefit from immunotherapeutic agents.

Materials and methods

Patients and samples

Tumor and control samples were obtained from 129patients with CRC and 121 patients with GC. The patientsunderwent surgical tumor resection at the ChanghaiHospital, Second Military Medical University, Shanghaiand Huadong Hospital Affiliated with Fudan University,Shanghai in 2013–2017. All samples were collected uponthe approval of the Ethics Committee of Academy ofForensic Science, Ministry of Justice, China. All subjectsprovided written informed consent.Tissue samples were obtained from resected tumors. The

relative percentage of tumor cells to nucleated cells wasassessed by a senior pathologist after hematoxylin andeosin staining. Samples with at least 30% tumor cells wereconsidered for further study. Peripheral blood cells andnormal intestinal tissues were used for control DNAisolation.

DNA preparation

Tissue DNAwas extracted from 10 formalin-fixed paraffin-embedded (FFPE) slides by using the QIAamp DNA FFPETissue Kit (Qiagen, Venlo, The Netherlands). Bloodcontrol DNA was extracted from 100 µL of peripheralblood by using the QIAamp DNA Blood Kit (Qiagen,Venlo, the Netherlands). All DNA was extracted inaccordance with the manufacturer’s instructions andquantified using a Qubit fluorometer (Life Technologies,Carlsbad, CA, USA). Extracted DNAwas stored at – 80 °Cuntil use.

Evaluation of MSI stability

Paired DNA were evaluated using a MSI Detection Kit(Microread, Beijing, China) for MSI stability. The assaycovered six quasimonomorphic mononucleotide repeats,

namely, BAT-25, BAT-26, NR-21, NR-22, NR-24, andMONO-27, two autosome short tandem repeats (STR;Penta C and Penta D), and a sex-related polymorphismAmel. The MSI status was evaluated in accordance withthe manufacturer’s instructions by comparing the matchingnormal and tumor sample pairs for shifts in allele sizes.Alternations in MSI status were divided into three

subgroups [19,20]: (1) MSI-H with the loci altered by morethan 30% (2/6); (2) MSS with stable MSI; and (3) MSI-Lcharacterized as the loci altered by less than 20% (1/6). Thealternations in MSI status were obtained and calculated forrespective samples (Fig. 1).

Evaluation of STR alternation status

The STR status was determined with a Goldeneye®20AForensic Identifier Kit (Peoplespot, Beijing, China)comprising 19 autosome STRs (13 CODIS core STRs,Penta E, Penta D, D2S1338, D19S433, D12S391, andD6S1043) and a sex-related polymorphism Amel. Fluor-escent multiplex polymerase chain reaction (PCR) wasused in accordance with the manufacturer’s protocol.Genotyping was performed in a 3100 ABI Prism GeneticAnalyzer (Applied Biosystems, USA) by using GeneMap-per Software (Applied Biosystems, USA), and subsequentanalyses excluded sex chromosome locus.Paired samples were investigated for the detection of

genotypes among the 19 somatic STRmarkers, which havebeen routinely used in forensic identification and kinship[21]. Against the control STR type, three types of STRalterations were determined and calculated for respectivesamples [22]: allelic loss (L), occurrence of an additionalallele (Aadd), and occurrence of a new allele (Anew)(Fig. 2). A sample was defined as L when a decreasedfluorescence signal in peak height (peak intensity ratio: thepeak ratio in the tumor tissue/corresponding peak ratio inthe normal tissues of < 0.5 or > 2) of an allele wasobserved after normal and tumor tissues were compared(Fig. 3).

Statistical analysis

All statistical analyses were performed using SPSS 19computer software (SPSS Chicago, IL, USA). All testswere two-tailed, and P values less than 0.05 wereconsidered statistically significant.

Results

Patients

Two-hundred fifty patients with GI tumors, 121 patientswith GC, and 129 patients with CRC, were diagnosed andresected in 2013–2017. The mean age of all patients at

102 Detecting genetic hypermutability of gastrointestinal tumor by a forensic STR kit

Fig. 1 MSI analysis of control and tumor DNA samples by using 6-monomorphic mononucleotide repeat loci markers, namely, NR-21, BAT-26,NR-27, BAT-25, NR-24, and MONO-27. (A) MSI positive sample (Sample No. 48). (B) MSI negative sample (Sample No. 237). The vertical andhorizontal axes refer to allele sizes and fluorescence intensity, respectively.

Anqi Chen et al. 103

diagnosis was 60.85 (range: 31–88) years, and 95 (38%)and 155 (62%) were females and males, respectively.

All MSI-H tumors can be observed alternations in STRgenotype

Matched MSI and STR assays were performed to all 250patients. Among these patients, 19 (7.6%) were observedwith instability in MSI (Supplementary Table S1). A totalof 15 MSI-H and 4 MSI-L were detected. A total of 9MSI-H and 3 MSI-L were isolated from 121 GC, and 6MSI-H and 1 MSI-L were isolated from 129 CRC (Table1). All the 19 MSI-positive patients were detected withvarying degrees of STR positive (range: 26.32%–84.21%)in the study (Table 2).

Allelic loss was the most common alternation in GIcancers

A total of 341 mutations were isolated from 121 GC tumortissues, and 411 mutations were isolated from 129 CRCtumor tissues. Among the 752 mutations, the occurrencesof L, Aadd, and Anew were 72.47% (545/752), 22.87%

(172/752), and 4.65% (35/752), respectively (Supplemen-tary Table S2). Allelic loss was the most frequently alteredmutation type among patients with GI. Aadd wasstatistically significantly high in patients with GC (P =0.0009), whereas Anew was high in patients with CRC (P= 0.002). No significant difference was found among theAnew between the patients with CRC and GC (P = 0.0968)(Table 3). We also discovered that the distribution of thealternations was quite different among the patients withdifferent MSI statuses, and that of allelic loss was commonin MSI-L/MSS samples (P < 0.0001). In MSI-H samples,Aadd was frequent statistically (P < 0.0001) (Table 3).

Mutation type in MSI-H and MSI-L/MSS was different

Exactly 235 samples with the status of MSI-L/MSS wereobserved in the study, and 590 mutations were found.Among the samples, 89.32% (527/590) exhibited allelicloss, 6.44% (38/590) was Aadd, and 4.24% (25/590) caseswere evaluated as Anew. The most frequently changed lociwere D18S51 (29.61%, 69/233), Penta E (20.26%,47/232), and CSF1PO (19.66%, 46/234) (SupplementaryTable S2).

Fig. 2 Electropherogram of the STR abnormalities. (A) Electropherogram of L at locus D8S1179 (Sample No. 130). Upper panel, reference DNAshowing a profile of alleles of 13 and 14; lower panel, partial loss of allele 14. (B) Electropherogram of Aadd at locus D16S539 (Sample No. 106).Upper panel, reference DNA showing a profile of alleles of 10 and 12; lower panel, additional allele 13. (C) Electropherogram of Anew at locusD18S51 (Sample No. 165). Upper panel, reference DNA showing a profile of alleles of 15 and 16; lower panel, new alleles of 13 and 14. The verticaland horizontal axes refer to allele sizes and fluorescence intensity, respectively.


Fig. 3 STR analysis of control and tumor DNA of an MSI-positive sample by using 19 autosomal STR markers (Sample No. 48). The vertical andhorizontal axes refer to allele sizes and fluorescence intensity, respectively.


In MSI-H samples, 162 mutations were isolated, and6.17% (10/162) was Anew. Only 11.11% (18/162) of themutation was classified as allelic loss, whereas 87.72%

(134/162) mutation was detected as Aadd (Table 3). Themost frequently changed loci among 15 MSI-H patientswere D18S51 (86.67%, 13/15), D8S1179 (80%, 12/15),and D3S1358 (73.33%, 11/15) (Supplementary Table S2).

Frequently altered MSI loci in GI tumors

All types of changes can be determined across the six loci.BAT-25 (7.2%, 18/250), BAT-26 (5.6%, 14/250), and NR-27(5.6%, 14/250) were the top three changes found amongthe patients with GI. All 15 MSI-H patients harbored themutations in BAT-25. BAT-25 was also the most frequentlyaltered locus in both CRC (100%, 7/7) and GC (91.67%,11/12) (Supplementary Table S1). In patients with CRC,the top three alternations were BAT-25 (5.43%, 7/129),BAT-26 (4.65%, 6/129), and NR-27 (4.65%, 6/129). In GCindividuals, the most frequently altered loci were BAT-25(9.09%, 11/121) and MONO-27 (7.44%, 9/121). Nosignificant difference was detected between the two typesof tumors (Table 4).

Frequently altered STR loci in GI tumors

The distribution of STR alternation incidence over the 19STR loci in all investigated GI cancer types is displayed inSupplementary Table S2. Patients with GI of 62.4%(156/250) displayed STR instability, and 68 patients(43.59%, 68/156) at 1–3 loci, 60 patients (38.46%, 60/156) at 4–7 loci, and 28 patients (17.95%, 28/156) at thenumber of loci more than 7 (Supplementary Table S3).In the study of 250 patients with GI, the most frequently

changed loci were D18S51 (32.79%), CSF1PO (22.09%),and Penta E (22.45%). The occurrence of allelic loss (L)was observed in 57.6% (144/250) of the patients with GI,and the most frequently changed loci were D18S51(26.32%), CSF1PO (18.47%), and Penta E (17.14%).Aadd occurred in 13.6% of the patients, and the mostfrequently altered loci were D19S433 (5.69%), D18S51(5.67%), and FGA (4.86%). Anew of 10.4% in the tumor

Table 2 STR status of all investigated MSI positive samples (n = 19)

Sample No.MSI STR

Mutation No. Status Mutation No. Alteration rate

021 6 MSI-H 15 78.95%

038 2 MSI-H 5 26.32%

043 1 MSI-L 9 47.37%

048 6 MSI-H 7 36.84%

056 6 MSI-H 16 84.21%

106 6 MSI-H 14 73.68%

108 5 MSI-H 10 55.56%

130 6 MSI-H 13 68.42%

135 6 MSI-H 10 52.63%

165 6 MSI-H 13 68.42%

168 1 MSI-L 10 52.63%

173 6 MSI-H 7 36.84%

178 1 MSI-L 14 73.68%

191 1 MSI-L 6 31.58%

207 5 MSI-H 10 52.63%

213 6 MSI-H 15 78.95%

223 4 MSI-H 8 42.11%

227 5 MSI-H 6 31.58%

236 6 MSI-H 13 68.42%

Table 1 Frequency of MSI in 250 gastrointestinal cancer samples

MSI statusCaner type

TotalGC (n = 121) CRC (n = 129)

MSS 109 122 231

MSI-L 3 1 4

MSI-H 9 6 15 (6%)

Total MSI 12 7 19

MSS, microsatellite stability; MSI-L, microsatellite instability low; MSI-H,microsatellite instability high; MSI, microsatellite instability.

Table 3 Difference of STR over the three types of alternation type in all investigated cancer types

STR alternation typeAlternation ratea

P valuecCRC GC

L 74.94% (308/411) 69.50% (237/341) 0.0968

Aadd 18.25% (75/411) 28.45% (97/341) 0.0009

Anew 6.81% (28/411) 2.05% (7/341) 0.002

STR alternation typeAlternation rateb

P valuecMSI-H MSI-L/MSS

L 11.11% (18/162) 89.32% (527/590) < 0.0001

Aadd 82.72% (134/162) 6.44% (38/590) < 0.0001

Anew 6.17% (10/162) 4.24% (25/590) 0.3009

a Difference of STR alternations in patients with CRC and GC.b Difference of the alternations between MSI-H and MSI-L/MSS patients.c Unpaired two-tailed t-test (P<0.05 statistically significant).


lesions replaced the one in control DNA, and the mostfrequently displayed loci were vWA (6.91%), Penta E(1.22%), and D18S51 (0.81%) (Table 5).

Discussion

MSI is a type of genomic instability caused by a defect inDNA mismatch repair (MMR) proteins, which are mainlyfound in the CRC and its hereditary form, hereditarynonpolyposis CRC [13,23–25]. MSI is characterized as theaccumulation of somatic alterations in the length of amicrosatellite [25] and a positive predictive factor with anopportunity for PD-1 inhibitor intervention [24]. However,Campanella et al. [23] reported that the characterization ofthe MSI phenotype is scarce, and is not a biomarker for theimmunotherapy response in GIST.

Given the abundance in the genome, STR markers arewidely applied in forensic science [21,22]; however, somegenomic regions contain genetic instability at certain lociby displaying allelic drop-out and/or multiple allele peaksin carcinogenesis [20]. STR status in tumor tissues aredifferent from corresponding normal controls, indicatingthat using polymorphic tetranucleotide repeats as markersfor forensic applications in tumor tissues is questionableand genotyping human DNA from the histopathologicaltissues must be carefully conducted [20,22,25]. Since thefailure of DNA replication leads to the changes in length ofalleles, resulting from insertion and/or deletion of repeatunits [26–28], that STRs can possibly measure geneticinstability and further be applied in the medical field.The current NCCN guidelines recommended MSI and

MMR testing in all patients with a personal history ofcolon or rectal cancer and MSI-H patients with highmutational burden. Treatment with immune checkpointinhibitor results in clinical benefits in MSI-H patients[20,24,29]. PD-L1 upregulation occurs in approximately30% of the patients with GC [10,13,24,28], and 30% of theCRCs have been established to respond to PD-1 inhibitors[30]. Meanwhile, only 5%–20% of the patients can beidentified using MSI testing [22,31,32]. The associationbetween tumor MSI and TMB has been evaluated [33], and21.9% (424/1934) MSS cases are TMB high, which maypredict the response of anti-PD1 therapy, and Hile et al.defined this form as MSI-A [14]. In addition to MSI, usingtargeted sequencing for the selected panel of genes candetect TMB [6,34]. However, the application was greatly

Table 4 Difference of MSI over the six loci in all investigated cancertypes

MSI lociAlternation rate

P valueCRC (n = 129) GC (n = 121)

NR-21 3.88% (5/129) 6.61% (8/121) 0.3322

BAT-26 4.65% (6/129) 6.61% (8/121) 0.5024

NR-27 4.65% (6/129) 6.61% (8/121) 0.5024

BAT-25 5.43% (7/129) 9.09% (11/121) 0.2644

NR-24 3.88% (5/129) 5.79% (7/121) 0.4824

MONO-27 3.10% (4/129) 7.44% (9/121) 0.1237

Unpaired two-tailed t-test (P < 0.05 statistically significant).

Table 5 STR alternations per locus in 250 GI samples

LocusSTR altered (%)

Totala L Aadd Anew

vWA 21.14% (52/246) 10.57% (26/246) 3.66% (9/246) 6.91% (17/246)

Penta E 22.45% (55/245) 17.14% (42/245) 4.08% (10/245) 1.22% (3/245)

D18S51 32.79% (81/247) 26.32% (65/247) 5.67% (14/247) 0.81% (2/247)

FGA 21.46% (53/247) 15.79% (39/247) 4.86% (12/247) 0.81% (2/247)

Penta D 16.8% (42/250) 13.2% (33/250) 2.8% (7/250) 0.8% (2/250)

D13S317 11.29% (28/248) 8.87% (22/248) 2.02% (5/248) 0.4% (1/248)

D6S1043 15.26% (38/249) 10.84% (27/249) 4.02% (10/249) 0.4% (1/249)

CSF1PO 22.09% (55/249) 18.47% (46/249) 3.61% (9/249) 0% (0/249)

D5S818 18.47% (46/249) 14.06% (35/249) 4.42% (11/249) 0% (0/249)

D8S1179 15.32% (38/248) 11.29% (28/248) 4.03% (10/248) 0% (0/248)

D12S391 14.17% (35/247) 11.34% (28/247) 2.83% (7/247) 0% (0/247)

D2S1338 14.06% (35/249) 10.84% (27/249) 3.21% (8/249) 0% (0/249)

D19S433 13.01% (32/246) 7.32% (18/246) 5.69% (14/246) 0% (0/246)

D21S11 12.45% (31/249) 7.63% (19/249) 4.82% (12/249) 0% (0/249)

D16S539 12% (30/250) 9.6% (24/250) 2.4% (6/250) 0% (0/250)

D7S820 11.65% (29/249) 7.23% (18/249) 4.42% (11/249) 0% (0/249)

D3S1358 10.8% (27/250) 6.8% (17/250) 4% (10/250) 0% (0/250)

TH01 8.87% (22/248) 7.66% (19/248) 1.21% (3/248) 0% (0/248)

TPOX 6.43% (16/249) 4.82% (12/249) 1.61% (4/249) 0% (0/249)

a Total number of alterations observed at each locus.


limited due to extremely high cost. Therefore, a biomarkerbetter than MSI status is necessary for the selection ofpatients who are in hypermutability and may benefit fromimmunotherapy.In our study, MSI is noted at markers BAT-26, NR-21,

BAT-25, NR-27, MONO-27, and NR-24. Patients with GIof 7.60% (19/250) possessed MSI alternations, including15 MSI-H (6%) and 4 MSI-L (1.6%). The MSI-Hincidence was 7.44% (9/121) and 4.65% (6/129) in GCand CRC, respectively (Table 1, Supplementary Table S 1),which is consistent with literature [31,32]. All the MSImarkers behaved fairly, and no statistically significantdifference was found in different GI cancers among the sixmarkers (Table 4). The highest rate of mutation was foundin BAT-25 (7.20%), followed by 14 cases for BAT-26(5.56%) and NR27 (5.56%). BAT-25 was also the mostfrequently altered marker in both patients with GC andCRC (Table 2, Supplementary Table S1). This outcomewas compatible with the results reported by Søreide [35]and Shemirani et al. [36]. All 15 MSI positive cases (9 GCand 6 CRC) showed the instability status in the STR test(Table 2), indicating the high accordance between theresults of MSI and STR.One of the contradictions in PD-1-based immunother-

apeutic treatment for GI cancers is the high-response rate(30%) and the low MSI-H detection rate (5%–20%)[22,30–32]. The main reason may be attributed to thenumber of markers we used. The number of loci used byMSI detection kit was usually five based on the NationalCancer Institute (NCI)-recommended panels of microsa-tellite markers [37], and the samples harboring 20% (1/5)and more than 40% (2/5) of the alternations were assessedas MSI-L and MSI-H, respectively. The disadvantages forthis system were self-evident because the evaluation resultsfor patients harboring 20%–40% of the mutation were stillambiguous, and this condition might be a major reason forthe contradiction mentioned above. To improve thepositive detection rate, finding another method fordetecting hypermutability is becoming an important issuethat must be solved.Laiho et al. [19] reported that all CRC have inherent

instability and some degree of MSI if enough markers aretested. Allelic loss is the most common molecular geneticalteration observed in human cancers, and the allelic losslevel is correlated with tumor progression [19]. Most of thepatients with CRC harbor widespread alternations in shortrepeated DNA sequences [38]. Therefore, similar to MSI,STR is a potential molecular marker for detecting changesin microsatellite allele length after Aadd, Anew, and allelicloss [20,25,38]. All MSI-H positive samples exhibitedSTR alternation, indicating that the STR testing in ourstudy might be a potential biomarker for screening genomeinstability except in MSI testing. To set the threshold foridentifying STR hypermutational status, we compared theresults of MSI and STR among 250 cases. Given that 30%

of patients with GI should respond to PD-1 blockade [30],75 out of 250 cases should then respond to PD-1 blockadein theory (Supplementary Table S3). Thus, the thresholdfor STR in the identification of hypermutational status is atleast 26.32% (5 out of 19). Among the 156 STR positiveGI individuals, 21.6% patients gained more than 6 loci,and 32% patients harbored at least 5 loci (SupplementaryTable S3). The result again indicated that the thresholdmight be 5 out of 19 (26.32%). Importantly, a total of 15MSI-H cases were found in this study, and STR statusranges from 84.21% (Sample 056) to 26.32% (Sample038). This result was consistent with the threshold(26.32%) we predicted.STRs are highly polymorphic microsatellite markers in

the human genome, which exhibits a high genetic diversityat population level due to allelic variations in the numberof repeat units of 2–5 base pairs [25]. When MMR wasdeficient, genetic instability is reflected in alternations inthe STR patterns [20]. The analysis of STRs is based on acapillary electrophoresis of PCR products from a com-mercial kit and not only provides an efficient and reliableway for identifying sample sources but also is useful indetecting genetic diseases and tumor genetic instability[25]. Our STR result showed a high mutation frequency intumor samples compared with its reference germlinemutation frequency (Table 6), indicating the widespreadSTR alternations in the tumor samples. Allelic loss andMSI are the common forms of genomic instability [25],and our study also indicated that allelic loss was the mostfrequently altered mutation types in GI tumors (Table 3).Thibodeau et al. [39] reported that allelic loss has been

demonstrated in a majority of colorectal tumors and isespecially altered in chromosomes 17p (≈75%), 18q(≈75%), and 5q (≈50%). In our study, chromosomes 18q(D18S51) and 5q (CSF1PO and D5S818) contributed26.32% (65/247) and 16.27% (81/498) allelic loss in 250patients with GI (Table 5). Although the percentage in thestudy (26.32% and 16.27%) differed considerably fromthat of Thibodeau et al. [39], chromosomes 18q and 5qalso contributed the largest mutation rate as previouslyreported. Thus, the results we provided were consistent,whereas the mutation rate was inconsistent. This incon-sistency in alternation rate might be attributed to thedifferent numbers of genetic alterations. We detected 19genetic alternations, whereas the number that Thibodeauet al. applied was only four. Chromosome 17p was notincluded in this study. Thus, the mutation rate contributedby chromosome 17p was not comparable. Nineteen STRloci were tested among 121 patients with GC and 129patients with CRC, and alternation frequency wasstatistically high in patients with CRC compared withpatients with GC in the loci D18S51 (P = 0.0007) andD5S818 (P = 0.0075) (Fig. 4). Allelic loss is dominant incolorectal tumors but is not a general characteristic of allcolorectal tumors [39].


Table 6 Characteristics of 19 STR loci

LocusChromosomallocation Repeat

Germline mutationfrequency [25,26]

Mutationfrequency

D19S433 19q12 (AAGG)(AAAG)(AAGG)(TAGG)[AAGG]n 0.11% 13.01% (32/246)

D7S820 7q21.11 [GATA] 0.10% 11.65% (29/249)

D6S1043 6q15 [AGAT]9–25 0.14% 15.26% (38/249)

CSF1PO 5q33.1 [AGAT] 0.16% 22.09% (55/249)

D5S818 5q23.2 [AGAT] 0.11% 18.47% (46/249)

FGA 4q28 [TTTC]3TTTTTTCT[CTTT]nCTCC[TTCC]2 0.28% 21.46% (53/247)

D3S1358 3p21.31 [AGAT], [TCTA] 0.12% 10.80% (27/250)

D2S1338 2q35 [TGCC]n[TTCC]n 0.12% 14.06% (35/249)

TPOX 2p25.3 [AATG] 0.01% 6.43% (16/249)

D21S11 21q21.1 [TCTA], [TCTG] 0.19% 12.45% (31/249)

Penta D 21q22.3 [AAAGA] 0.14% 16.80% (42/250)

D18S51 18q21.33 [GAAA] 0.22% 32.79% (81/247)

D16S539 16q24.1 [GATA] 0.11% 12.00% (30/250)

Penta E 15q26.2 [AAAGA] 0.16% 22.45% (55/245)

D13S317 13q31.1 [TATC] 0.14% 11.29% (28/248)

D12S391 12p13.2 [AGAT]8–17[AGAC]6–10[AGAT]0–1 0.24% 14.17% (35/247)

Vwa 12p13.31 [TCTA] with [TCTG] and [TCCA] inserts 0.17% 21.14% (52/246)

TH01 11p15.5 [TCAT] 0.01% 8.87% (22/248)

D8S1179 8q24.13 [TATC] 0.14% 15.32% (38/248)

Fig. 4 Frequency of genetic instabilities at each STR locus.


The classification of MSI is based on the NCI guidelines[40]. In the application of the 6-locus panel of the MSImarkers, tumor samples harboring more than one alterna-tion (33.33%) were classified as MSI-H, and onealternation (16.67%) was classified as MSI-L. If moremarkers were used, the MSI-H group was defined astumors containing 20% or more MSI, whereas the MSI-Ltumors exhibit 20% or less MSI. The approach determin-ing the STR status as genome instability status mightovercome the problems inherent to the original five- or six-marker panel. This method might facilitate the widespreadscreening for hypermutability in tumors of patients with GIcancers.Owing to the growing understanding on tumor signaling

and mechanism at the molecular level, the strategies in thetreatment of GI tumor are improving. Traditional non-selective chemotherapy is gradually being augmented.Thus, the detection rate of these abnormalities must beimproved for clinical screening. However, detecting tumorburden by comparing STR status is a method of choice thatmust be further studied and verified.

Acknowledgements

This work was supported by the grants from National Key Researchand Development Program of China (Nos. 2016YFA0500600 and

2016YFC0800703), the National Science Fund for DistinguishedYoung Scholars (No. 81625013), and General Program of NationalNatural Science Foundation of China (No. 81772028). The funders

had no role in study design, data analysis, publishing decisions, ormanuscript preparation.

Compliance with ethics guidelines

Anqi Chen, Suhua Zhang, Jixi Li, Chaoneng Ji, Jinzhong Chen, andChengtao Li declare that they have no conflicts of interest. All

conducted procedures were in accordance with the ethical standardsof the human laboratory responsibility committee (institutions andcountries).

Electronic Supplementary Material Supplementary material isavailable in the online version of this article at http://dx.doi.org/

10.1007/s11684-019-0698-4 and is accessible for authorized users.

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detecting genetic hypermutability of gastrointestinal

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