Study population and sample collection
The present study included 19 newly diagnosed fresh surgical tumor specimens from 19 CRC patients attending to the outpatient clinic of our department. All patients were subjected to surgical resection where the surgeon removed the part of the colon that contains the tumor mass, along with a margin of normal tissue on either side of the mass. After gross examination, macro dissection was done. Then, hematoxylin and eosin (H&E) staining was performed on serial sections. The tissue area representing the “tumor” which contains the highest numbers of cancer cells was identified and “normal” which contains no malignant tissue identified and separated. Healthy control tissue was included in our study for the interpretation of MSI results.
Patients were selected according to the following criteria:
Inclusion criteria:
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Age group: adulthood CRC patients
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Gender: males and females
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Pathologically proved colorectal adenocarcinoma.
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Stages II, III, and IV
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Newly diagnosed cases attending the outpatient clinic
Exclusion criteria:
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Patients with double malignancy
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Known HIV-positive or AIDS-related illness
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Women who are pregnant or breastfeeding
Our study was exploratory in nature and performed in the molecular lab of our institution, in the period from March 2018 to February 2019. The study was approved by our department Institutional Review Board (IRB)-3-2018. All procedures performed in the study involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments (GCP guidelines) or comparable ethical standards.
DNA extraction
Genomic DNA was extracted from tissues with the QIAamp DNA Mini kit (Qiagen, USA) as reported by the manufacturer and was eluted in 60 μl volume. The extracted DNA samples were measured utilizing the Qubit dsDNA High Sensitivity (HS) assay kit (Life Technologies, Fisher Scientific, USA). A quantitative PCR (qPCR) reaction was performed to conclude the amplifiability of the extracted gDNA samples and to predict assay success. All samples with ΔCq value below or equal to 2 can be selected for further use.
Library preparation
Libraries were prepared using TruSight Tumor 15 kit according to the manufacturer’s protocol (Illumina, Inc., San Diego, CA). TruSight Tumor 15 uses next-generation sequencing (NGS) to assess 15 of the most commonly mutated significant genes in CRC in a single assay: AKT serine/threonine kinase 1 (AKT1), G protein subunit alpha 11 (GNA11), NRAS, BRAF, G protein subunit alpha q (GNAQ), platelet-derived growth factor receptor alpha (PDGFRA), EGFR, KIT proto-oncogene receptor tyrosine kinase (KIT), PIK3CA, erb-b2 receptor tyrosine kinase 2 (ERBB2), KRAS, ret. proto-oncogene (RET), forkhead box L2 (FOXL2), MET proto-oncogene, receptor tyrosine kinase (MET), and TP53. Library quality was checked out by 2100 Bioanalyzer utilizing the Bioanalyzer DNA 1000 reagents and chips (Agilent Technologies, Santa Clara, CA). Successful library amplification was estimated when the expected PCR product size is ~ 350 bp. Before sequencing, the libraries together with PhiX control library were normalized following the manufacturer’s protocol, and equal volumes were pooled to constitute the terminal sequencing library. The TruSight Tumor 15 sequencing panel achieves limits of detection of 5% variant allele frequency across 250 amplicons with 93.5% of bases covered at ≥ 500× [9].
Sequencing and data analysis
Sequencing was accomplished utilizing MiSeqDx device (Illumina) with a 2 × 151 bp read length and a total time of 27 h which involve cluster generation, sequencing, and base calling on the MiSeqDx system. Specifications based on Illumina PhiX control library support cluster densities between 1200 and 1400 k/mm2 clusters passing filter for v3 chemistry. The quality scores > 80% bases higher than Q30 at 2 × 151 bp. Image processing and VCF file generation were further analyzed; we then annotated the variants using VariantStudio™ software version 3. This software determined the numerical identifier for each variant in the COSMIC database, if the genomic variant position overlapped a variant recorded in COSMIC. The COSMIC ID linked to the COSMIC page associated with the identifier to determine information on whether that variant has been detected before in any cancer patient or a novel one. Synonymous variants and non-coding regions were filtered out. Mutations with low depth, which indicate ≤ 50× depths, were filtered out. And mutations with ≤5% variant allele frequency were filtered out. Variant quality which is one parameter of the variant call format (VCF) < 80% was filtered out.
MSI analysis
We evaluated the MSI status using three mononucleotide repeat markers (BAT25, BAT26, and NR27). The kit was purchased from Qiagen Co., with primer sequences as follows:
BAT-25 (product size, 114 bp): 5′-CTCGCCTCCAAGAATGTAAGT-3′; 5′-CTATGGCTCTAAAATGCTCTGTTC-3′
BAT-26 (product size, 122 bp): 5′-TGACTACTTTTGACTTCAGCC-3′; 5′-AACCATTCAACATTTTTAACCC-3′
NR-27 (product size, 89 bp): 5′-AACCATGCTTGCAAACCACT-3′; 5′-CGATAATACTAGCAATGACC-3′.
Amplification reactions (20 μl) were prepared with 10–100 ng extracted DNA, 1× PCR buffer, 1.5 mM MgCl2, 0.25 mM dNTP, 0.5 μM of each primer, and 0.5 units Platinum® Taq DNA Polymerase (Invitrogen). Reactions were subjected to PCR amplification: initial incubation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 54 °C for 45 s, and 72 °C for 60 s, and a final incubation at 72 °C for 10 min. PCR products were analyzed on the Bioanalyzer 2100 system. The results were evaluated by comparing every tumor DNA to DNA from the healthy control tissue. Peaks present in the tumor tissue which were not found in the normal tissue indicated instability of a marker. Samples with no altered markers were classified as MSS. Samples with only one altered microsatellite marker were classified as MSI-L. Samples with ≥ 2 altered markers were classified as MSI-H [10].
Statistical analysis
Statistical analysis was done using IBM© SPSS© Statistics version 22 (IBM© Corp., Armonk, NY, USA). Numerical data were expressed as mean and standard deviation or median and range as appropriate. Qualitative data were expressed as frequency and percentage. A chi-square test was used to examine the relation between the qualitative variables. For not normally distributed quantitative data, a comparison between the two groups was done using the Mann-Whitney test (non-parametric t test). All tests were two-tailed. A p value ≤ 0.05 was considered significant.
