Diagnostic value of the BRAF variant V595E in urine samples, smears and biopsies from canine transitional cell carcinoma
Diagnostic value of the BRAF variant V595E in urine samples, smears and biopsies from canine transitional cell carcinoma
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DOI：
10.15654/tpk-180554
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发表时间：
2018-10-01
影响因子：
0.6
通讯作者：
Pantke, Peter
中科院分区：
文献类型：
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作者：
Aupperle-Lellbach, Heike;Grassinger, Julia;Pantke, Peter
目的：移行细胞癌（transitional cell carcinoma，TCC）是犬泌尿道最常见的恶性肿瘤.此前，通过对冷冻和石蜡包埋组织切片以及尿液中的TCC肿瘤细胞进行DNA测序，在约85%的犬TCC病例中鉴定出BRAF基因V595 E突变。本研究的目的是在细胞学涂片中建立这些方法，并调查我们患者队列中犬TCC中BRAF突变V595 E的患病率。材料和方法：选择来自66只患有TCC（n = 33）、膀胱息肉（n = 7）、膀胱炎（n = 23）或无膀胱疾病（n = 3）的狗的活检样品（n = 43）、尿液（n = 48）和/或细胞学涂片（n = 31），提交用于常规诊断。使用市售试剂盒从石蜡材料、尿液和细胞学涂片中分离DNA。通过桑格测序检查外显子15中BRAF突变c.1784 T>A的存在。结果：在43份石蜡包埋活检标本中有39份和48份尿样中有38份分离到足够量的高质量DNA。DNA分离和测序在16/18个细胞计数高的涂片中成功，但在10/13个细胞密度低的涂片中不成功。在可获得不同样本材料的所有病例中，石蜡包埋组织、细胞学涂片和/或尿液的BRAF分析结果相同。在患有TCC的22/31只犬（70.9%）中，证实了BRAF突变的存在，而在没有病理学发现或患有膀胱炎或息肉的动物中无法检测到BRAF突变。结论和临床意义：BRAF突变分析是一种新的和良好的方法，能够在不确定的情况下大多数确定TCC的诊断。非侵入性诊断样品，包括含有足够数量的相关细胞的尿液和尿沉淀物以及细胞学抽吸物和福尔马林固定的活检物，可用于分析。然而，重要的是要注意，只有阳性鉴定的突变是诊断。进一步的研究是必要的，以调查预后和治疗相关的变异，以及如何这种遗传分析可以作为一种早期检测方法TCC。
Objective: Transitional cell carcinoma (TCC) is the most common malignant tumour of the canine urinary tract. Previously, the mutation of the BRAF gene V595E was identified in approximately 85% of canine TCC cases by DNA sequencing of TCC tumour cells, both in frozen and paraffin-embedded tissue sections, as well as in urine. The objective of this study was to establish these methods in cytological smears and to investigate the prevalence of BRAF mutation V595E in canine TCC in our cohort of patients. Material and methods: Biopsy samples (n = 43), urine (n = 48) and/or cytological smears (n = 31) from 66 dogs with TCC (n = 33), urinary bladder polyps (n = 7), cystitis (n = 23) or without bladder diseases (n = 3), submitted for routine diagnostics, were selected. DNA isolation from paraffin material, urine and cytological smears was performed using commercially available kits. Exon 15 was examined for the presence of the BRAF mutation c.1784T>A by Sanger sequencing. Results: In 39/43 paraffin-embedded biopsies and 38/48 urine samples, a sufficient amount of good quality DNA was isolated. DNA isolation and sequencing were successful in 16/18 smears with a high cell count, but not in the 10/13 smears with low cellularity. In all cases from which different sample materials were available, the results of BRAF analysis were identical in paraffin-embedded tissue, cytological smears and/or urine. In 22/31 dogs (70.9%) with TCC, the presence of the BRAF mutation was confirmed, whereas it could not be detected in animals without pathological findings or with cystitis or with a polyp. Conclusion and clinical relevance: BRAF mutation analysis is a new and good method to be able to mostly confirm a diagnosis of TCC in uncertain cases. Non-invasive diagnostic samples, including urine and urine sediment containing sufficient numbers of relevant cells as well as cytology aspirates and formalin-fixed biopsies can be used for analysis. However, it is important to note that only a positive identification of the mutation is diagnostic. Further research is necessary to investigate prognostic and therapeutic relevance of the variant and how this genetic analysis can be used as an early detection method for TCC.