Bratisl Lek Listy. 2022;123(10):758-768. doi: 10.4149/BLL_2022_122.
The main aim of the study was to evaluate the potential roles of KRAS/NRAS proto-oncogenes, IL-4 VNTR variants and HPV prevalence in colorectal cancer metastasis. As the second aim, the interactions of the analyzed genes and viral sequences with both clinicopathological variables and each other were targeted.
DNA was extracted using AmoyDx FFPE DNA Extraction kit from paraffin-embedded colorectal tumor tissue samples (n = 60). NRAS/KRAS mutational profiles were determined with real-time polymerase chain reaction using AmoyDx KRAS/NRAS Mutation Detection Kit. Genotyping of IL-4 VNTR was made with PCR. HPV detection was analyzed by PCR with both GP5+/GP6+ consensus primers and type-specific primers for HPV-16 and HPV-18. SPSS v22 (IBM) statistics software was used for all statistical analyses.
From the demographical/clinicopathological parameters, age and biopsy specimens revealed an association with metastasis. KRAS mutation rate was as high as 65 % in the patients and the most prevalent mutation type was G12D. Metastasis risk was 3.19-fold increased in KRAS-mutated patients compared to KRAS-negative ones. IL-4 VNTR genotypes/alleles were not associated with metastasis in our analysis. The frequency of HPVs in our colorectal cancer cohort was 36.7 %, but HPV positivity was not found to be associated with metastasis. A significant association was found between HPV and NRAS mutations; NRAS wild-type status acted as a protective factor by 7.5-fold for HPV negativity.
Our study comprehensively and concomitantly evaluated several potential molecular risk factors. Future studies designed in such combined approaches will substantially contribute to better manage colorectal cancer tumorigenesis from molecular biological perspective (Tab. 6, Fig. 2, Ref. 40).
本研究的主要目的是评估 KRAS/NRAS 原癌基因、IL-4VNTR 变体和 HPV 流行率在结直肠癌转移中的潜在作用。作为第二个目的，分析了这些基因和病毒序列与临床病理变量及彼此之间的相互作用。
采用 AmoyDxFFPE DNA 提取试剂盒从石蜡包埋的结直肠肿瘤组织样本（n=60）中提取 DNA。采用 AmoyDxKRAS/NRAS 突变检测试剂盒实时聚合酶链反应（PCR）检测 NRAS/KRAS 突变谱。采用 PCR 对 IL-4VNTR 进行基因分型。采用 GP5+/GP6+ 通用引物和 HPV-16、HPV-18 型特异性引物的 PCR 法检测 HPV。采用 SPSSv22（IBM）统计软件进行所有统计分析。
从人口统计学/临床病理参数来看，年龄和活检标本与转移有关。患者的 KRAS 突变率高达 65%，最常见的突变类型是 G12D。与 KRAS 阴性患者相比，KRAS 突变患者的转移风险增加了 3.19 倍。在我们的分析中，IL-4VNTR 基因型/等位基因与转移无关。我们结直肠癌队列中 HPV 的频率为 36.7%，但 HPV 阳性与转移无关。我们发现 HPV 与 NRAS 突变之间存在显著相关性；NRAS 野生型状态使 HPV 阴性的保护作用增加了 7.5 倍。
本研究全面、同时评估了几种潜在的分子危险因素。未来设计的此类联合方法的研究将从分子生物学角度为更好地管理结直肠癌肿瘤发生做出实质性贡献（表 6、图 2、参考文献 40）。
