Microsatellite instability test using peptide nucleic acid probe-mediated melting point analysis: a comparison study

Cell lines and patient tissue samples

For testing the sensitivity of PNA (U-TOP™ MSI Detection Kit) and MNR (Promega MSI Analysis System Kit), HeLa (microsatellite stable, MSS) and SNU-638 (MSI-H) cell lines were used. HeLa cells and SNU-638 cells were cultured with DMEM and RPMI1640 medium, respectively. Each medium was supplemented with 10% FBS (Invitrogen Life Technologies, Carlsbad, CA, USA) and 1% penicillin/streptomycin.

Formalin-fixed paraffin-embedded (FFPE) tissue samples were collected from 2263 patients with CRCs who visited Severance Hospital between January 2005 and December 2015. A total of 166 CRCs (76 diagnosed as MSI-H and 90 diagnosed as MSS) and matched non-neoplastic colon mucosal tissues were randomly selected for this study. MSI status was previously analyzed using the NCI method. The specimens were obtained from the archives of the Department of Pathology, Yonsei University, Seoul, Korea and the Liver Cancer Specimen Bank of the National Research Resource Bank Program of the Korean Science and Engineering Foundation, Ministry of Science and Technology.

PNA probe-mediated real-time PCR sensing for detection of MSI status

We tested the performance of the PNA method in detecting MSI status in colon cancer samples using genomic DNA samples (gDNAs) extracted from FFPE CRCs and matched normal tissues. gDNA was isolated according to the manufacturer’s instructions (QIAamp DNA FFPE Tissue Kit, Qiagen, Venlo, Netherlands; Maxwell® 16 FFPE Purification Kit for DNA, Promega). Both quality and quantity of extracted genomic DNA samples were evaluated by using the Nanodrop (Thermo Waltham, MA, USA) and subsequent gel electrophoresis. In PNA method, wild type PNA probe perfectly hybridized with wild type allele, while partial or mismatch hybridization caused melting temperature of PNA probe to be lower than that of perfectly matched probe. At denaturation temperature, PNA probe was subject to fluorescence quenching by random coiling, and this quenching temperature was analyzed by real-time PCR machine [9, 10].

To assess MSI status with PNA method, U-TOP™ MSI Detection Kit was purchased from SeaSun Biomaterials (Daejeon, Korea). This commercially available product employed five MSI marker genes (NR21, NR24, NR27, BAT25, and BAT26). A 20-μl mixture composed of gDNA sample (3 μl), 2 × qPCR Premix (10 μl), and dual-labeled (fluorescence and quencher) PNA probes for NR21, NR24, and BAT26 (MSI Marker A; 7 μl), as well as another 20-μl mixture composed of gDNA sample (3 μl), 2 × qPCR Premix (10 μl) and dual-labeled PNA probes for BAT25 and NR27 (MSI Marker B; 7 μl). The qPCR premix contained dNTP and DNA Taq polymerase, as well as Uracil DNA Glycosylase (UDG). Each PNA probe was fluorescently labeled with Texas-Red, Hexachloro-fluorescein, or Fluorescein amidite. Two individual real-time PCR reactions were performed for each normal and cancer sample using a CFX96 PCR machine (Bio-Rad, Hercules, CA, USA). PCR reaction steps consisted of amplification and subsequent melting point analysis. The amplification condition was 50 °C for 5 min, 95 °C for 10 min, and 60 cycles of 95 °C for 30 s, 65 °C for 30 s, 55 °C for 30 s, and 57 °C for 45 s. The initial incubation at 50 °C for 5 min was required to activate Uracil DNA Glycosylase and prevent possible carryover contamination. In 60 cycles of PCR, four different temperatures and respective optimal times were required. The main purpose of using these conditions was to ensure specific binding of PNA probes to their target MSI marker. In detail, 95 °C for 30 s was for denaturation of template DNAs, 65 °C for 30 s was for binding of PNA probes to their target MSI markers, 55 °C for 30 s was for annealing temperature of primers, and 57 °C for 45 s was for elongation of a polymerase. The melting point analysis condition was 10 min at 95 °C for denaturation and touchdown PCR (90 °C to 45 °C, decreasing 1 °C per cycle). Fluorescence was measured for 10 s at each cycle of touchdown PCR. Obtained melting peaks were analyzed to detect alterations in the five MSI marker genes. A CRC sample was considered to be unstable in a MSI marker gene when a > 3 °C melting temperature difference between a CRC and the normal sample was observed. For the analysis of minimal base pair alteration that PNA method can detect, we used artificially synthesized oligo targets using either deletions or insertions (Macrogen, Seoul, Korea).

PCR fragment analysis

For NCI method, five microsatellite loci (BAT-25, BAT-26, D2S123, D5S346, and D17S250) recommended by the 1997 NCI-sponsored MSI workshop were amplified in a single multiplex PCR reaction. PCR products were analyzed by capillary electrophoresis. For interpretation, instability at more than one locus was defined as MSI-H, instability at a single locus was defined as low MSI (MSI-L), and no instability at any locus was defined as MSS. For MNR method, amplification of five MSI markers (NR21, NR24, BAT26, BAT25, and NR27) was performed using gDNAs extracted from CRCs and matched normal samples. PCR reactions were performed with the MSI Analysis System Kit referring to the MSI Analysis System Version 1.2 protocol. This kit was purchased from Promega (Madison, WI, USA). Amplified PCR products were purified using a PCR clean-up kit (Macherey-Nagel, Düren, Germany), and the size of PCR amplicons was analyzed using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Based on data obtained from the sequencer, MSI status was determined by random experts (Macrogen).

Immunohistochemistry

Paraffin-embedded tissue blocks were cut into 4-μm sections. IHC analysis was performed using a Ventana XT automated stainer (Ventana Corporation, Tucson, AZ, USA) with antibodies against the following: MutL homolog 1 (MLH1, diluted 1:50, BD Biosciences, San Jose, CA, USA), MutS homolog 2 (MSH2, diluted 1:200, BD Biosciences), MutS homolog 6 (MSH6, diluted 1:100, Cell Marque, Rocklin, CA, USA), and PMS1 homolog 2 (PMS2, diluted 1:40, Cell Marque). Positive internal controls including stromal cells and lymphoid cells were confirmed, and the percentage of nuclear expression was measured.

Statistical analysis

To calculate the diagnostic sensitivity and specificity of each MSI test, McNemar’s tests were performed. The sensitivity (%) of each MSI analysis method was calculated as follows: 100 × true positives/(true positives + false negatives), where true positives and false negatives were defined according to MSI status originally diagnosed by NCI method. The specificity (%) of PNA method was calculated as follows: 100 × true negatives/(false positives + true negatives), where true negatives and false positives were defined according to MSI status originally diagnosed by NCI method.

P = average PPV and NPV values from reference articles; P₀= expected PPV and NPV for this study (equivalence); Z_α/2=1.96; Z_β = 0.842.

Based on the calculation above, we came to the conclusion that more than 68 MSI-H and 89 MSS CRC samples were required for this study, and performed MSI analysis in 76 MSI-H and 90 MSS CRC samples.

To analyze clinicopathological parameters, statistical analyses were performed using SPSS software, version 21.0.0.0 for Windows (IBM., Armonk, NY, USA). Mann-Whitney tests, Student’s t-tests, Fisher’s exact tests, or χ² tests were used depending on the purpose; P-values < 0.05 were considered statistically significant.

Determination of MSI status by three conventional MSI detection methods

We randomly collected 166 cases from 2263 CRCs that had undergone MSI status analysis with variable methods from January 2005 to December 2015. The NCI analysis results of the 166 cases were also collected. Seventy-six cases were previously diagnosed as MSI-H and 90 cases as MSS or MSI-L.

To test the performance of conventional MSI detection methods, we conducted MNR method with five quasi-monomorphic mononucleotide markers (Promega MSI Analysis System Kit) and IHC with four MMR proteins (MLH1, MSH2, MSH6, and PMS2). MSI analysis results determined by each method are summarized in Additional file 1: Table S1. Among the 76 MSI-H cases, 74 cases showed MSI-H in all three conventional MSI tests. Case no. 1 was diagnosed as MSI-H by NCI and IHC but was diagnosed as MSI-L by MNR. Case no. 20 was diagnosed as MSI-H by NCI and MNR, but no loss of protein expression was detected by IHC (Additional file 1: Table S1 and Additional file 2: Figure S1).

Diagnostic sensitivity and specificity of three MSI test methods

Next, we evaluated the maximum sensitivity of the MNR and PNA methods, which used the same five MSI markers. To do so, mixed samples of gDNAs extracted from HeLa cells (microsatellite stable, MSS) and SNU-638 cells (MSI-H) were used, and MNR and PNA analyses were performed with different proportions (0, 1, 5, 10, 20, 40, and 100%) of MSI-H variant. MNR analysis showed that SNU-638 cells harbored 7-, 8-, 8-, 12-, and 10-base deletion mutations in NR21, NR24, BAT25, BAT26, and NR27, respectively. PNA method was capable of detecting alterations in all five MSI markers in the mixed samples, including down to 5% MSI-H variant, whereas MNR method required a sample containing up to 20% MSI-H variant to detect alterations in all five MSI markers. PNA method was further capable of detecting 1% MSI-H variant in NR21 and BAT25 markers (Fig. 1 and Additional file 3: Table S2). The repeatedly performed experiments using PNA method provided consistent and reproducible results, irrespective of the type of MSI markers and portion of MSI variants. To evaluate the qualitative performance of PNA method, we determined the minimum alteration number in each MSI marker that PNA method can detect, using artificially synthesized oligo targets containing one or two-base deletion mutations and one or two-base insertion mutations. The samples used in this experiment were composed of 100% MSI variant. Oligo targets containing insertion mutations were included because mononucleotide microsatellites often exhibit insertion mutations. The analysis result showed that PNA method clearly detected up to two-base deletion and one-base insertion mutations of the five MSI markers (Additional file 2: Figure S2). Then, we performed PNA analysis using titrated samples with MSI variants containing a two-base deletion or one-base insertion to evaluate the quantitative performance of PNA method. The analysis results showed that PNA method clearly detected a two-base deletion or one-base insertion of the five MSI markers in samples containing more than 5% MSI-H variants (Fig. 2).

Determination of MSI status by the PNA probe-mediated melting point analysis

MSI status was determined by PNA method, which was a recently developed real-time PCR based method. The results of PNA method showed sharp melting curves highly specific to each cancer sample as compared with paired normal samples (Fig. 3, left panel). MNR method also showed readable size differences between cancer and paired normal samples (Fig. 3, right panel). Samples with alterations in more than one MSI marker were determined as MSI-H, whereas samples with an alteration in a single MSI marker or no alteration were determined as MSI-L or MSS, respectively (Fig. 3 and Additional file 2: Figure S3). MSI-L and MSS were grouped together for statistical analysis based on a previous report of no significant clinicopathological or molecular differences between MSI-L and MSS CRCs [21]. MSI analysis using PNA method indicated that 76 samples were MSI-H and the remaining 90 were MSS, which was the same result as that of NCI analysis. A comparison of instability in each MSI marker between PNA and MNR method showed significant difference in NR24, with PNA having higher detection rates (Additional file 3: Table S3). We also note that one case (Case no. 1) was determined as MSI-L by MNR but was determined as MSI-H by NCI, IHC, and PNA methods (Additional file 1: Table S1).

Clinicopathologic findings of sporadic and hereditary MSI-H CRCs

We observed that MSI-H and MSS CRCs showed distinctive clinicopathologic characteristics, as previously reported [2]. Compared with MSS CRCs, MSI-H CRCs were more frequently found in the right colon and showed larger and exophytic features. Elevated pre-operative carcinoembryonic antigen (CEA) level and advanced T stage were noted in MSS CRCs. Mucinous adenocarcinoma and signet ring cell carcinoma were observed more often in MSI-H CRCs than in MSS CRCs. Patients with MSI-H CRCs were younger on average than patients with MSS CRCs (Additional file 3: Table S4).

Among the 166 CRCs, 155 cases did not satisfy Amsterdam II and Revised Bethesda criteria, which allowed us to clinically define these cases as sporadic CRCs. Eleven cases showed clinical features of HNPCC as suggested by Amsterdam II or Revised Bethesda criteria. Analysis of these 11 clinically diagnosed HNPCC cases by NCI, MNR, IHC, and PNA methods showed that two cases were determined as MSS in all four tests, indicating that these two cases can be considered as familial colorectal cancer type X. In the remaining nine cases, MSI-H was determined by all four tests and mutations in MLH1 or MSH2 were detected (data not shown), which leads us to consider the nine cases are Lynch syndrome. Consequently, nine and 67 cases were classified as MSI-H CRCs associated with Lynch syndrome and sporadic MSI-H CRCs, respectively. Whereas most clinicopathologic characteristics of the nine cases with Lynch syndrome and 67 sporadic MSI-H CRC cases were similar, some parameters showed significant differences (Additional file 3: Table S5). Patients in the Lynch syndrome group were younger on average than patients in the sporadic CRC group. Also, mean tumor size was smaller in the Lynch syndrome group, perhaps because the features of hereditary CRCs, such as family history and early symptoms, may lead to early health check-ups and the detection of smaller tumors. There were no differences in other parameters such as MMR protein expression status, tumor location, pre-operative CEA level, gross type, histologic diagnosis, T stage, lymphovascular invasion, mucin formation, Crohn-like reaction, or tumor budding.

Diagnostic value of IHC using antibodies against four MMR proteins to determine MSI status

Next, we performed IHC analysis of four MMR proteins in the 166 CRCs to determine MSI status. MSI-H was defined as the loss of expression of at least one MMR protein in more than 95% of tumor cells. By this criterion, we detected MSI-H in 75 out of 76 samples (98.7%). The percentage of nuclear expression of MMR proteins was also measured in all cases, and MSI status was further analyzed according to various cut-off values of loss of MMR protein expression (Additional file 3: Table S6). When we defined MMR deficit as a complete loss of nuclear staining, we observed 84.21% concordance with the originally diagnosed MSI status using two antibodies against MLH1 and MSH2, 73.68% concordance with two antibodies against PMS2 and MSH6, and 90.79% concordance with all four antibodies against MLH1, MSH2, PMS2, and MSH6. When we defined MMR deficit as < 1% nuclear staining, we observed 84.21% concordance with MLH1 and MSH2 antibodies, 89.47% concordance with PMS2 and MSH6 antibodies, and 96.05% with all four antibodies. When we defined MMR as < 5% nuclear staining, we observed 90.79% concordance with MLH1 and MSH2 antibodies and 98.68% concordance with PMS2 and MSH6 antibodies or all four antibodies. Overall, therefore, an IHC criterion of 95% was the best match to the original MSI diagnosis results.