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

Abstract

Growing evidence suggests that somatic hypermutational status and programmed cell death-1 overexpression are potential predictive biomarkers indicating treatment benefits from immunotherapy using immune checkpoint inhibitors. However, biomarker-matched trials are still limited, and many of the genomic alterations remain difficult to target. To isolate the potential somatic hypermutational tumor from microsatellite instability low/microsatellite stability (MSI-L/MSS) cases, we employed two commercial kits to determine MSI and forensic short tandem repeat (STR) alternations in 250 gastrointestinal (GI) tumors. Three types of forensic STR alternations, namely, allelic loss, Aadd, and Anew, were identified. 62.4% (156/250) of the patients with GI exhibited STR alternation, including 100% (15/15) and 60% (141/235) of the microsatellite high instability and MSI-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 and suggested that it might be a potential threshold. Given the similar mechanism of the mutations of MSI and forensic STR, the widely used forensic identifier STR kit might provide potential usage for identifying hypermutational status 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 DNA repeat region hypermutation and instability [¹]. Multiple tests are available to evaluate MMR-D status [²]. These methods include immunohistochemistry staining for mismatch repair proteins, MLH1 promoter methylation analysis, and microsatellite instability (MSI) evaluation [³–⁶]. These tests exhibit prognostic and therapeutic implications for patients with colorectal carcinoma and gastric and endometrial cancers [⁷]. In MSI, cancer produces frame-shift mutations, yielding abnormal proteins and novel epitopes [⁸]. The accumulation of mutations causes the development of high antigenicity immune phenotype that may respond to immune checkpoint inhibitors. Approximately 5%–21% of colorectal cancers (CRCs) or/and gastric cancers are characterized by different MSI methods [¹,⁹,¹⁰]. Most MSI high (MSI-H) and/or PD-L1 positive cases respond to immune checkpoint inhibitors, whereas some MSI low/microsatellite stability (MSI-L/MSS) cases still respond to the agent [¹¹–¹³]. This phenomenon suggests the existence of alternative MSI forms (A-MSI) [¹⁴,¹⁵].

Future tumor research with immune therapeutic approaches will possibly focus on identifying strategies to improve non-MSI-H tumor immunogenicity. Elevated hypermutation alterations with improved MSI detection methods, such as elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) and next-generation sequencing (NGS)-based tumor mutation burden (TMB), can identify some hypermutation cases from the MSI-L group [¹⁶–¹⁸]. These methods may improve the biomarker-matched trials and increase the number of patients that benefit from immunotherapy with immune checkpoint inhibitors. However, EMAST application was limited by the lack of a consensus criterion, whereas the TMB measured by NGS was time- and cost-defective. Therefore, generating a rapid and convenient method for identifying additional A-MSI in patients with tumor is urgently needed.

Herein, we reported a study combined with two commercial kits to determine the hypermutation status in numerous patients with gastrointestinal (GI) cancer to provide a new potential biomarker to detect patients with genetic hypermutability in addition to assess the MSI analysis. This process may help screen patients who can benefit from immunotherapeutic agents.

Materials and methods

Patients and samples

Tumor and control samples were obtained from 129 patients with CRC and 121 patients with GC. The patients underwent surgical tumor resection at the Changhai Hospital, Second Military Medical University, Shanghai and Huadong Hospital Affiliated with Fudan University, Shanghai in 2013–2017. All samples were collected upon the approval of the Ethics Committee of Academy of Forensic Science, Ministry of Justice, China. All subjects provided written informed consent.

Tissue samples were obtained from resected tumors. The relative percentage of tumor cells to nucleated cells was assessed by a senior pathologist after hematoxylin and eosin staining. Samples with at least 30% tumor cells were considered for further study. Peripheral blood cells and normal intestinal tissues were used for control DNA isolation.

DNA preparation

Tissue DNA was extracted from 10 formalin-fixed paraffin-embedded (FFPE) slides by using the QIAamp DNA FFPE Tissue Kit (Qiagen, Venlo, The Netherlands). Blood control DNA was extracted from 100 µL of peripheral blood by using the QIAamp DNA Blood Kit (Qiagen, Venlo, the Netherlands). All DNA was extracted in accordance with the manufacturer’s instructions and quantified using a Qubit fluorometer (Life Technologies, Carlsbad, CA, USA). Extracted DNA was stored at −80 °C until use.

Evaluation of MSI stability

Paired DNA were evaluated using a MSI Detection Kit (Microread, Beijing, China) for MSI stability. The assay covered six quasimonomorphic mononucleotide repeats, namely, BAT-25, BAT-26, NR-21, NR-22, NR-24, and MONO-27, two autosome short tandem repeats (STR; Penta C and Penta D), and a sex-related polymorphism Amel. The MSI status was evaluated in accordance with the manufacturer’s instructions by comparing the matching normal 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 more than 30% (2/6); (2) MSS with stable MSI; and (3) MSI-L characterized as the loci altered by less than 20% (1/6). The alternations in MSI status were obtained and calculated for respective samples (Fig. 1).

Evaluation of STR alternation status

The STR status was determined with a Goldeneye^®20A Forensic Identifier Kit (Peoplespot, Beijing, China) comprising 19 autosome STRs (13 CODIS core STRs, Penta E, Penta D, D2S1338, D19S433, D12S391, and D6S1043) and a sex-related polymorphism Amel. Fluorescent multiplex polymerase chain reaction (PCR) was used in accordance with the manufacturer’s protocol. Genotyping was performed in a 3100 ABI Prism Genetic Analyzer (Applied Biosystems, USA) by using GeneMapper Software (Applied Biosystems, USA), and subsequent analyses excluded sex chromosome locus.

Paired samples were investigated for the detection of genotypes among the 19 somatic STR markers, which have been routinely used in forensic identification and kinship [²¹]. Against the control STR type, three types of STR alterations were determined and calculated for respective samples [²²]: allelic loss (L), occurrence of an additional allele (Aadd), and occurrence of a new allele (Anew) (Fig. 2). A sample was defined as L when a decreased fluorescence signal in peak height (peak intensity ratio: the peak ratio in the tumor tissue/corresponding peak ratio in the normal tissues of<0.5 or>2) of an allele was observed after normal and tumor tissues were compared (Fig. 3).

Statistical analysis

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

Results

Patients

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

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

Allelic loss was the most common alternation in GI cancers

A total of 341 mutations were isolated from 121 GC tumor tissues, and 411 mutations were isolated from 129 CRC tumor tissues. Among the 752 mutations, the occurrences of L, Aadd, and Anew were 72.47% (545/752), 22.87% (172/752), and 4.65% (35/752), respectively (Supplementary Table S2). Allelic loss was the most frequently altered mutation type among patients with GI. Aadd was statistically 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 the Anew between the patients with CRC and GC (P = 0.0968) (Table 3). We also discovered that the distribution of the alternations was quite different among the patients with different MSI statuses, and that of allelic loss was common in 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 were observed in the study, and 590 mutations were found. Among the samples, 89.32% (527/590) exhibited allelic loss, 6.44% (38/590) was Aadd, and 4.24% (25/590) cases were evaluated as Anew. The most frequently changed loci were D18S51 (29.61%, 69/233), Penta E (20.26%, 47/232), and CSF1PO (19.66%, 46/234) (Supplementary Table S2).

In MSI-H samples, 162 mutations were isolated, and 6.17% (10/162) was Anew. Only 11.11% (18/162) of the mutation was classified as allelic loss, whereas 87.72% (134/162) mutation was detected as Aadd (Table 3). The most frequently changed loci among 15 MSI-H patients were 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 among the patients with GI. All 15 MSI-H patients harbored the mutations in BAT-25. BAT-25 was also the most frequently altered 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 GC individuals, the most frequently altered loci were BAT-25 (9.09%, 11/121) and MONO-27 (7.44%, 9/121). No significant difference was detected between the two types of tumors (Table 4).

Frequently altered STR loci in GI tumors

The distribution of STR alternation incidence over the 19 STR loci in all investigated GI cancer types is displayed in Supplementary 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 the number 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 most frequently altered loci were D19S433 (5.69%), D18S51 (5.67%), and FGA (4.86%). Anew of 10.4% in the tumor lesions replaced the one in control DNA, and the most frequently 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 in DNA mismatch repair (MMR) proteins, which are mainly found in the CRC and its hereditary form, hereditary nonpolyposis CRC [¹³,²³–²⁵]. MSI is characterized as the accumulation of somatic alterations in the length of a microsatellite [²⁵] and a positive predictive factor with an opportunity for PD-1 inhibitor intervention [²⁴]. However, Campanella et al. [²³] reported that the characterization of the MSI phenotype is scarce, and is not a biomarker for the immunotherapy response in GIST.

Given the abundance in the genome, STR markers are widely applied in forensic science [²¹,²²]; however, some genomic regions contain genetic instability at certain loci by displaying allelic drop-out and/or multiple allele peaks in carcinogenesis [²⁰]. STR status in tumor tissues are different from corresponding normal controls, indicating that using polymorphic tetranucleotide repeats as markers for forensic applications in tumor tissues is questionable and genotyping human DNA from the histopathological tissues must be carefully conducted [²⁰,²²,²⁵]. Since the failure of DNA replication leads to the changes in length of alleles, resulting from insertion and/or deletion of repeat units [²⁶–²⁸], that STRs can possibly measure genetic instability and further be applied in the medical field.

The current NCCN guidelines recommended MSI and MMR testing in all patients with a personal history of colon or rectal cancer and MSI-H patients with high mutational burden. Treatment with immune checkpoint inhibitor results in clinical benefits in MSI-H patients [²⁰,²⁴,²⁹]. PD-L1 upregulation occurs in approximately 30% of the patients with GC [¹⁰,¹³,²⁴,²⁸], and 30% of the CRCs have been established to respond to PD-1 inhibitors [³⁰]. Meanwhile, only 5%–20% of the patients can be identified using MSI testing [²²,³¹,³²]. The association between tumor MSI and TMB has been evaluated [³³], and 21.9% (424/1934) MSS cases are TMB high, which may predict the response of anti-PD1 therapy, and Hile et al. defined this form as MSI-A [¹⁴]. In addition to MSI, using targeted sequencing for the selected panel of genes can detect TMB [⁶,³⁴]. However, the application was greatly limited due to extremely high cost. Therefore, a biomarker better than MSI status is necessary for the selection of patients who are in hypermutability and may benefit from immunotherapy.

In our study, MSI is noted at markers BAT-26, NR-21, BAT-25, NR-27, MONO-27, and NR-24. Patients with GI of 7.60% (19/250) possessed MSI alternations, including 15 MSI-H (6%) and 4 MSI-L (1.6%). The MSI-H incidence was 7.44% (9/121) and 4.65% (6/129) in GC and CRC, respectively (Table 1, Supplementary Table S 1), which is consistent with literature [³¹,³²]. All the MSI markers behaved fairly, and no statistically significant difference was found in different GI cancers among the six markers (Table 4). The highest rate of mutation was found in BAT-25 (7.20%), followed by 14 cases for BAT-26 (5.56%) and NR27 (5.56%). BAT-25 was also the most frequently altered marker in both patients with GC and CRC (Table 2, Supplementary Table S1). This outcome was compatible with the results reported by Søreide [³⁵] and Shemirani et al. [³⁶]. All 15 MSI positive cases (9 GC and 6 CRC) showed the instability status in the STR test (Table 2), indicating the high accordance between the results of MSI and STR.

One of the contradictions in PD-1-based immunotherapeutic 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 the number of markers we used. The number of loci used by MSI detection kit was usually five based on the National Cancer Institute (NCI)-recommended panels of microsatellite markers [³⁷], and the samples harboring 20% (1/5) and more than 40% (2/5) of the alternations were assessed as MSI-L and MSI-H, respectively. The disadvantages for this system were self-evident because the evaluation results for patients harboring 20%–40% of the mutation were still ambiguous, and this condition might be a major reason for the contradiction mentioned above. To improve the positive detection rate, finding another method for detecting hypermutability is becoming an important issue that must be solved.

Laiho et al. [¹⁹] reported that all CRC have inherent instability and some degree of MSI if enough markers are tested. Allelic loss is the most common molecular genetic alteration observed in human cancers, and the allelic loss level is correlated with tumor progression [¹⁹]. Most of the patients with CRC harbor widespread alternations in short repeated DNA sequences [³⁸]. Therefore, similar to MSI, STR is a potential molecular marker for detecting changes in microsatellite allele length after Aadd, Anew, and allelic loss [²⁰,²⁵,³⁸]. All MSI-H positive samples exhibited STR alternation, indicating that the STR testing in our study might be a potential biomarker for screening genome instability except in MSI testing. To set the threshold for identifying STR hypermutational status, we compared the results 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 blockade in theory (Supplementary Table S3). Thus, the threshold for STR in the identification of hypermutational status is at least 26.32% (5 out of 19). Among the 156 STR positive GI individuals, 21.6% patients gained more than 6 loci, and 32% patients harbored at least 5 loci (Supplementary Table S3). The result again indicated that the threshold might be 5 out of 19 (26.32%). Importantly, a total of 15 MSI-H cases were found in this study, and STR status ranges from 84.21% (Sample 056) to 26.32% (Sample 038). 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 diversity at population level due to allelic variations in the number of repeat units of 2–5 base pairs [²⁵]. When MMR was deficient, genetic instability is reflected in alternations in the STR patterns [²⁰]. The analysis of STRs is based on a capillary electrophoresis of PCR products from a commercial kit and not only provides an efficient and reliable way for identifying sample sources but also is useful in detecting genetic diseases and tumor genetic instability [²⁵]. Our STR result showed a high mutation frequency in tumor samples compared with its reference germline mutation frequency (Table 6), indicating the widespread STR alternations in the tumor samples. Allelic loss and MSI are the common forms of genomic instability [²⁵], and our study also indicated that allelic loss was the most frequently 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 is especially altered in chromosomes 17p (≈75%), 18q (≈75%), and 5q (≈50%). In our study, chromosomes 18q (D18S51) and 5q (CSF1PO and D5S818) contributed 26.32% (65/247) and 16.27% (81/498) allelic loss in 250 patients with GI (Table 5). Although the percentage in the study (26.32% and 16.27%) differed considerably from that of Thibodeau et al. [³⁹], chromosomes 18q and 5q also contributed the largest mutation rate as previously reported. Thus, the results we provided were consistent, whereas the mutation rate was inconsistent. This inconsistency in alternation rate might be attributed to the different numbers of genetic alterations. We detected 19 genetic alternations, whereas the number that Thibodeau et al. applied was only four. Chromosome 17p was not included in this study. Thus, the mutation rate contributed by chromosome 17p was not comparable. Nineteen STR loci were tested among 121 patients with GC and 129 patients with CRC, and alternation frequency was statistically high in patients with CRC compared with patients with GC in the loci D18S51 (P = 0.0007) and D5S818 (P = 0.0075) (Fig. 4). Allelic loss is dominant in colorectal tumors but is not a general characteristic of all colorectal tumors [³⁹].

The classification of MSI is based on the NCI guidelines [⁴⁰]. In the application of the 6-locus panel of the MSI markers, tumor samples harboring more than one alternation (33.33%) were classified as MSI-H, and one alternation (16.67%) was classified as MSI-L. If more markers were used, the MSI-H group was defined as tumors containing 20% or more MSI, whereas the MSI-L tumors exhibit 20% or less MSI. The approach determining the STR status as genome instability status might overcome the problems inherent to the original five- or six-marker panel. This method might facilitate the widespread screening for hypermutability in tumors of patients with GI cancers.

Owing to the growing understanding on tumor signaling and mechanism at the molecular level, the strategies in the treatment of GI tumor are improving. Traditional nonselective chemotherapy is gradually being augmented. Thus, the detection rate of these abnormalities must be improved for clinical screening. However, detecting tumor burden by comparing STR status is a method of choice that must be further studied and verified.

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