Patient
We enrolled in this study hundred patients with primary colon cancer resected between 2105 and 2020, at Hassan II University Hospital, Fez, Morocco. The medical charts were prospectively and retrospectively reviewed and patients were included according to the following inclusion criterion: patients with histologically confirmed primary adenocarcinoma, all cases with I-IV stage colon cancer, and patients with prognostic data. Patients were excluded from this study due to the following exclusion criteria: All patients with incomplete clinical records, patients without histological confirmation of colon adenocarcinoma, and patients with rectal cancer.
Demographic and clinicopathological data (e.g. age, gender, tumor grade, tumor localization histological subtype, tumor grade, disease stage, and numbers of examined regional lymph nodes…) and follow up data, were collected from the patient’s medical records and pathology reports.
Assessment of tumor budding
Tumor budding was assessed on hematoxylin-eosin and Safran (HES) stained slides. For each CC case, one representative HES slide was used for scoring by the pathologist according to the ITBCC recommendations. Tumor buds were evaluated in a single hotspot measuring 0.785mm2 at the invasive front using microscopy at 20 × objectives.
We then used a three-tier system which is recommended by the ITBCC group to provide tumor budding count and tumor budding category. The system of scoring is categorized as:
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0–4 buds—low budding (Bd 1)
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5–9 buds—intermediate budding (Bd 2)
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10 or more buds—high budding (Bd 3)
We grouped the patients to be low-intermediate (grade1 + grade2) and high tumor budding (grade 3).
Determination of Mismatch repair protein status
The immunohistochemistry (IHC) method was used to establish the mismatch repair tumor status (MSS or MSI) and to detect the intact or the loss expression of the MMR proteins (MLH1, PMS2, MSH2, and MSH6). The IHC study was performed on unstained formalin-fixed paraffin-embedded (FFPE) tumor tissue sections of 5 µm thickness, on the automated immunostainer Ventana Benchmark ULTRA. We have employed monoclonal antibodies specific for each MMR protein, MLH1 (G168-728/CELL MARQUE), MSH2 (G219-1129/CELL MARQUE), MSH6 (44/CELL MARQUE), and PMS2 (MRQ-28/CELL MARQUE).Adjacent normal tissue (lymphocytes or normal glandular cells) was used as an internal control for positive staining.
Detection of KRAS and NRAS mutation
DNA extraction
Genomic DNA was extracted from 5–8 sections of 5 µm thickness of macro-dissected formalin-fixed paraffin-embedded (FFPE) tumor blocks, containing at least 50% of tumor cells, as determined by an experienced pathologist on H&E-stained paraffin slides. The extraction was effected using the QIAamp DNA FFPE Tissue Kit (Invitrogen), and according to the manufacturer’s instructions. DNA concentration (ng/ul) was assessed by Qubit fluorometer.
PCR and direct sequencing
For each sample, mutations of KRAS exons 2, 3 and 4, and NRAS exons 2 and 3, were amplified by polymerase chain reaction (PCR). Briefly, 10 ng of template DNA was amplified using, 12× PCR mix platinium, 12.5 pmol primers, 50 umol Mgcl2 and 2.5 ul of the corresponding set of PCR primers listed in Table 1. After the purification of PCR products, the presence of mutations was detected by direct sequencing using the BigDye Terminator V3.1 Cycle Sequencing Kit (ABI Prism) and the Applied Biosystems 3500Dx Genetic Analyzer (Applied Biosystem).
Table 1
Primer sequences used for PCR.
Primer name | Primer sequence |
KRAS-ex 2- F KRAS-ex 2- R | 5’-GGTGGAGTATTTGATAGTGTA- 3’ 5’-TGCATATTACTGGTGCAGACC- 3’ |
KRAS-ex 3- F KRAS-ex 3- R | 5’-AGTAAAAGGTGCACTGTAATAA-3’ 5’-ATAATAAGCTGACATTAAGGAG-3’ |
KRAS-ex 4- F KRAS-ex 4- R | 5’-TGTTACTAATGACTGTGCTATAACTTTT-3’ 5’-TATGCTATACTATACTAGGAAATAAAA-3’ |
NRAS-ex2-F NRAS-ex2-R | 5’-ATGACTGAGTACAAACTGGTGGTGGTTGGAGCA-3’ 5’-CACTTTGTAGATGAATATGATCCCACCATAGAG-3’ |
NRAS-ex3-F NRAS-ex3-R | 5’-GATTCTTACAGAAAACAAGTGGTTA-3’ 5’-CATTTGCGGATATTAACCTCTACAG-3’ |
NRAS-ex4-F NRAS-ex4-R | 5’-GGAGCAGATTAAGCGAG-3’ 5’-TCAGCCAAGACCAGACAG-3’ |
Pyrosequencing
The analysis of RAS mutations was performed using the TheraScreen® KRAS Pyro kit (for KRAS codons 12 and 13), and the TheraScreen® RAS Extension Pyro kit (for KRAS codons 59/61, 117 and 146, and NRAS codons 12, 13, 59, 61, 117 and 146) (Qiagen, GERMANY), according to the manufacturer's instructions. As described previously(6), 5 µl of template DNA (2–10 ng of genomic DNA) were amplified by polymerase chain reaction (PCR) in a 20 µl volume containing 12.5 µl of PyroMark® PCR Master Mix 2x, 2.5 µl of Coral Load Concentrate 10x, 4 µl of nuclease-free water, and 1 µl of the corresponding set of PCR primers (Qiagen). 10 µl of PCR products were immobilized to Streptavidin Sepharose High Performance beads (Qiagen) to prepare the single‑stranded DNA. The corresponding sequencing primers were allowed to anneal to the DNA using a PyroMark Q24 plate and a vacuum workstation (Qiagen). PyroMark Q24 reagents (enzyme mixture, substrate mixture and nucleotideall from Qiagen) were prepared and loaded into a cartridge in order to be dispensed during the sequencing process. Finally, the sequences were analyzed using Pyromark Q24 software in the AQ analysis mode. In each run, two controls were included: negative control (without template DNA) and an unmethylated control DNA, provided by the kit as a positive control for PCR and sequencing reactions were included.
Statistical analysis
Clinical, pathological, and molecular variables collected at baseline were described as means and standard deviation (sd’s) for quantitative variables and percentages for qualitative variables. Associations between tumor budding (assessing as a categorical variable) and categorical factors of tumor were assessed using the χ2-test or Fisher’s exact test variables. The unpaired t-test was used for continuous variables. Tests were statistically significant when p < 0.05.
Overall survival was defined as the time from the start of diagnosis until death or until the last follow-up. Relapse-free survival was measured from the date of initial diagnosis until the date of local relapse or regional relapse or last follow-up/death (all causes) whichever occurs first.
RFS and OS rates according to tumor budding, clinicopathological factors, and molecular features were determined using the Kaplan‑Meier method, and survival differences between groups were evaluated by log-rank test.
Multivariate analysis was performed using a Cox proportional hazard model to identify independent risk factors for survival. Factors that were significant and nearly significant in univariate analysis (P < 0.1) were included in multivariate analysis.
Data from univariate and multivariate analyses were reported as hazard ratios (HRs) with 95% confidence intervals (CIs). All statistics were assessed using 2-sided tests, with P values < .05 considered statistically significant. Statistical analysis was performed using the IBM SPSS Statistic 21.