METHODS: Seventy-two FFPE samples, including 36 control samples (19 vulva;17 anal) for threshold setting and 36 samples (24 vulva; 12 anal) for clinical evaluation, were analyzed by mFAST-SeqS. CNA in vulva and anal lesions were determined by calculating genome-wide and chromosome arm-specific z-scores in comparison with the respective control samples. Sixteen samples were also analyzed with the conventional Shallow-seq approach.
RESULTS: Genome-wide z-scores increased with the severity of disease, with highest values being found in cancers. In vulva samples median and inter quartile ranges [IQR] were 1[0-2] in normal tissues (n = 4), 3[1-7] in premalignant lesions (n = 9) and 21[13-48] in cancers (n = 10). In anal samples, median [IQR] were 0[0-1] in normal tissues (n = 4), 14[6-38] in premalignant lesions (n = 4) and 18[9-31] in cancers (n = 4). At threshold 4, all controls were CNA negative, while 8/13 premalignant lesions and 12/14 cancers were CNA positive. CNA captured by mFAST-SeqS were mostly also found by Shallow-seq.
CONCLUSIONS: mFAST-SeqS is easy to perform, requires less DNA and less sequencing reads reducing costs, thereby providing a good alternative for Shallow-seq to determine CNA in small FFPE samples.
方法:72个FFPE样品,包括36个对照样本(19个外阴;17个肛门)用于阈值设定和36个样本(24个外阴;12个肛门)用于临床评估,通过mFAST-SeqS进行分析。通过与各自的对照样品进行比较,计算全基因组和染色体臂特异性z评分,确定外阴和肛门病变中的CNA。还用常规Shallow-seq方法分析了16个样品。
结果:全基因组z值随着疾病的严重程度而增加,在癌症中发现的价值最高。在外阴样本中,正常组织(n=4)的中位数和四分位数间范围[IQR]为1[0-2],3[1-7]在癌前病变(n=9)和21[13-48]在癌症(n=10)。在肛门样本中,正常组织中的中位数[IQR]为0[0-1](n=4),14[6-38]在癌前病变(n=4)和18[9-31]在癌症(n=4)。在阈值4时,所有对照均为CNA阴性,而8/13的癌前病变和12/14的癌症为CNA阳性。mFAST-SeqS捕获的CNA大多也被Shallow-seq发现。
结论:mFAST-SeqS易于执行,需要更少的DNA和更少的测序读数,降低成本,从而为Shallow-seq确定小FFPE样品中的CNA提供了良好的替代方案。