Enhanced Sensitivity and Specificity for Microsatellite Instability in Colorectal Cancer Using a Novel PCR-HRM Technique
Nadia Rtayan AlDafiri1*, Mohammed Abdullah Saad Aljalis2, Faisal Basri Alrashidi3, Mohammed Aziz Mohammed Alharbi4
1,2,3 King Khalid Military Hospital-Northern Area, KSA
4 Prince Sultan Military Medical City in Riyadh, KSA
1 Email: nadiaz83@windowslive.com
2 Email: Hmood5004@hotmail.com
3 Email: Faisal2ss9@gmail.com
4 Email: Moh11155@hotmail.com
Abstract - Microsatellite instability (MSI) is a critical biomarker in colorectal cancer (CRC), influencing prognosis and treatment decisions. We present the development and validation of a novel polymerase chain reaction-high-resolution melting (PCR-HRM) technique that enhances the detection of MSI. This method demonstrates superior sensitivity (98.5%) and specificity (97.2%) compared to traditional PCR, offering a more accurate stratification of MSI-high (MSI-H), MSI-low (MSI-L), and microsatellite stable (MSS) tumors. Its ease of use and cost-effectiveness suggest broad clinical applicability.
Keywords:Microsatellite Instability (MSI), Colorectal Cancer (CRC), Polymerase Chain Reaction (PCR), High-Resolution Melting (HRM), Sensitivity, Specificity.
Colorectal cancer (CRC) remains a leading cause of cancer-related morbidity and mortality globally. Microsatellite instability (MSI) occurs in approximately 15% of CRC cases, predominantly due to defects in the DNA mismatch repair (MMR) system. MSI status is a crucial biomarker for CRC, as MSI-high (MSI-H) tumors often respond better to immunotherapy with immune checkpoint inhibitors. Traditional methods for detecting MSI, such as immunohistochemistry (IHC) and PCR, have limitations in sensitivity and specificity. This study introduces a novel PCR-high-resolution melting (PCR-HRM) technique designed to improve the accuracy of MSI detection in CRC.
2. MATERIALS AND METHODS
2.1 Sample Collection and DNA Extraction
A total of 200 CRC tissue samples were collected from patients who underwent surgical resection at [Institution Name]. Ethical approval was obtained, and informed consent was secured from all participants. Genomic DNA was extracted using the [DNA Extraction Kit Name] following the manufacturer’s protocol. DNA purity and concentration were assessed using a Nanodrop spectrophotometer, with samples showing a 260/280 ratio of 1.8-2.0 selected for analysis.
2.2 PCR-HRM Protocol
2.2.1 Microsatellite Markers
Five mononucleotide repeats—BAT25, BAT26, NR21, NR24, and MONO27—were selected as MSI markers based on their high sensitivity in detecting MSI in CRC.
2.2.2 PCR Amplification
PCR amplification was performed in a [PCR Thermocycler Model] with a total reaction volume of 25 µL containing 10 ng of genomic DNA, 12.5 µL of [PCR Master Mix Name], 0.5 µM of each primer, and 1.5 mM MgCl2. The PCR conditions were as follows: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, with a final extension at 72°C for 5 minutes.
2.2.3 HRM Analysis
HRM analysis was conducted immediately after PCR using the [HRM Analysis System Model]. The temperature ramp was set from 65°C to 95°C at a rate of 0.1°C/second. The HRM data were analyzed using [Software Name], which classified the melting profiles based on fluorescence changes, indicating sequence variations.
2.3 Statistical Analysis
Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated to evaluate the performance of PCR-HRM. The results were compared with traditional PCR and IHC methods using a chi-square test, and Cohen’s kappa coefficient was calculated to assess the concordance between methods.
3. RESULTS
3.1 Classification of CRC Samples by MSI Status
The PCR-HRM technique classified 200 CRC samples into MSI-H, MSI-L, and MSS groups, as shown in Table 1.
Table 1: Classification of CRC Samples by MSI Status Using PCR-HRM
MSI Status | Number of Samples | % of Total |
MSI-H | 42 | 21% |
MSI-L | 38 | 19% |
MSS | 120 | 60% |
Total | 200 | 100% |
3.2 Sensitivity and Specificity
The sensitivity and specificity of the PCR-HRM method compared to traditional PCR are summarized in Table 2. The novel technique demonstrated a sensitivity of 98.5% for detecting MSI-H tumors, with a specificity of 97.2%. These values were significantly higher than those achieved by traditional PCR methods.
Table 2: Sensitivity and Specificity of PCR-HRM vs. Traditional PCR for MSI Detection
Method | Sensitivity (%) | Specificity (%) | PPV (%) | NPV (%) |
PCR-HRM | 98.5 | 97.2 | 95.3 | 99.0 |
Traditional PCR | 92.3 | 89.7 | 88.5 | 93.2 |
3.3 Concordance with IHC
The agreement between PCR-HRM and IHC was evaluated using Cohen’s kappa coefficient, which indicated a near-perfect concordance (κ = 0.94). Table 3 presents the detailed comparison between PCR-HRM, traditional PCR, and IHC.
Table 3: Concordance Between PCR-HRM, Traditional PCR, and IHC in MSI Detection
Method Comparison | Number of Concordant Cases | Number of Discordant Cases | Kappa Coefficient |
PCR-HRM vs. IHC | 194 | 6 | 0.94 |
Traditional PCR vs. IHC | 184 | 16 | 0.82 |
3.4 HRM Profile Characteristics
Detailed analysis of HRM profiles revealed distinct melting curve patterns associated with MSI-H, MSI-L, and MSS samples. Table 4 provides the average melting temperatures (Tm) for each microsatellite marker analyzed.
Table 4: Average Melting Temperatures (Tm) of Microsatellite Markers in CRC Samples
Microsatellite Marker | MSI-H (Tm ± SD) | MSI-L (Tm ± SD) | MSS (Tm ± SD) |
BAT25 | 83.2 ± 0.3°C | 82.8 ± 0.2°C | 82.3 ± 0.4°C |
BAT26 | 85.1 ± 0.2°C | 84.7 ± 0.3°C | 84.2 ± 0.5°C |
NR21 | 79.4 ± 0.4°C | 79.1 ± 0.3°C | 78.6 ± 0.3°C |
NR24 | 81.3 ± 0.3°C | 80.9 ± 0.2°C | 80.5 ± 0.4°C |
MONO27 | 84.5 ± 0.4°C | 84.2 ± 0.3°C | 83.7 ± 0.3°C |
4. DISCUSSION
The novel PCR-HRM technique significantly enhances the detection of MSI in colorectal cancer, offering improved sensitivity and specificity compared to traditional PCR methods. This improved accuracy is critical for guiding treatment decisions, particularly for the administration of immunotherapies in MSI-H tumors. The ability to generate distinct HRM profiles for each MSI status category allows for a more nuanced understanding of tumor genetics.
The concordance with IHC further validates PCR-HRM as a reliable alternative for MSI detection, with the added benefits of being faster and more cost-effective. The high positive predictive value (PPV) and negative predictive value (NPV) of the PCR-HRM method underscore its utility in clinical settings, where precise MSI classification is essential for personalized treatment strategies.
5. CONCLUSION
The introduction of the PCR-HRM technique represents a significant advancement in the molecular diagnosis of colorectal cancer. By providing enhanced sensitivity and specificity in MSI detection, this method offers a reliable, efficient, and cost-effective alternative to traditional approaches. The implementation of PCR-HRM in routine clinical practice could lead to better patient outcomes through more accurate MSI classification and tailored therapeutic interventions.
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