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.
To determine the sample number required for this study, we performed non-inferiority test at a significance level of 0.05 and a statistical power of 80%. The average of positive predictive value (PPV) and negative predictive value (NPV) were obtained from 10 reference articles [11,12,13,14,15,16,17,18,19,20], where methods using microsatellite instability testing or real-time PCR were compared to immunohistochemistry for detecting mutations of MLH1, MSH2, MSH6, and PMS2 or other human genes. The average PPV and NPV were 91.1 and 93.6%, respectively, and the differences from lower margin of 95% confidence intervals (CI; δ, non-inferiority margin) were − 5.49% and − 4.3%, respectively. Based on reference articles, the sizes of MSI-H and MSS samples were calculated using the equation below, considering a 10% dropout rate.
$$ \mathrm{N}=\frac{{\left({Z}_{\alpha /2}+{Z}_{\beta}\right)}^2P\left(1-P\right)\ }{{\left(\delta -\left|P-{P}_0\right|\right)}^2} $$
P = average PPV and NPV values from reference articles; P0= expected PPV and NPV for this study (equivalence); Zα/2=1.96; Zβ = 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.
