Computational protocol: CA-SSR1 Polymorphism in Intron 1 of the EGFR Gene in Patients with Malignant Tumors Who Develop Acneiform Rash Associated with the Use of Cetuximab

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Protocol publication

[…] For the genotype analysis, DNA was isolated from peripheral blood by using the Blood Mini kit (A&A Biotechnology, Gdynia, Poland). The isolated DNA was re-suspended in 100 µL of TE buffer and stored at −20 °C until further analysis.To determine the degree of polymorphism in the CA-SSR1 region, a PCR reaction was carried out, followed by genotyping with the use of the GenScan method. The primers used in the PCR were designed by using Primer3 on the basis of the sequence obtained from the National Center for Biotechnology Information (NCBI) database (reference sequence: NG_007726). The length of the PCR product was 298 bp, with 16 repetitions of the CA dinucleotide. To analyze the GenScan, one primer was end-labeled with the fluorescent dye FAM (forward primer 5-FAMGGACTCTTGAGCGGAAGC-3 and reverse primer 5-CCATAAACCCACTTGACAGG-3). The PCR reaction was performed under the following conditions: 3 min initial denaturation at 94 °C, followed by 30 cycles of denaturation for 60 s at 94 °C, annealing for 60 s at 61 °C, and elongation for 60 s at 72 °C; the final extension was carried out for 7 min at 72 °C.The fragment length was determined by analysis carried out with the GeneScan ABI Prism® 3700 DNA Analyzer (Life Technologies, Carlsbad, CA, USA). GS500 ROX (-250 LIZ) size marker was added to each sample. To validate the first method, GenScan direct sequencing was carried out for several randomly selected homozygote samples. [...] Statistical analysis was performed with the Statistica software version 10 (Statsoft, Inc., 2011; http://www.statsoft.com). Due to the relatively small group of patients and the lack of normal distributions in the studied variables, we used non-parametric statistical methods.The clinical response to cetuximab [Response Evaluation Criteria In Solid Tumors (RECIST)] was determined for 49 patients. The association between response to treatment and rash or genotype was determined by using Spearman’s rank correlation. The association between rash severity (NCI CTCAE v3.0) and CA-SSR1 genotype subgroup [cutoffs: S ≤ 17(CA), L > 17(CA), n(CA) ≤ 35, and n(CA) > 35] was determined using Pearson’s chi-square test. The relationship between body surface area covered by rash and CA-SSR1 genotype was determined by using Spearman’s rank correlation, Mann Whitney U test, and Kruskal–Wallis test. Two quantitative variables, the percentage of body surface area covered by rash and the sum of CA dinucleotide repeats in the EGRF gene, were analyzed using Pearson’s correlation. The significance level for all tests was p < 0.05. […]

Pipeline specifications

Software tools GENSCAN, Primer3, Statistica
Applications Miscellaneous, qPCR
Organisms Homo sapiens
Diseases Exanthema, Neoplasms