Abstract：
BACKGROUND: Shallow whole genome sequencing (Shallow-seq) is used to determine the copy number aberrations (CNA) in tissue samples and circulating tumor DNA. However, costs of NGS and challenges of small biopsies ask for an alternative to the untargeted NGS approaches. The mFAST-SeqS approach, relying on LINE-1 repeat amplification, showed a good correlation with Shallow-seq to detect CNA in blood samples. In the present study, we evaluated whether mFAST-SeqS is suitable to assess CNA in small formalin-fixed paraffin-embedded (FFPE) tissue specimens, using vulva and anal HPV-related lesions.
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.
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.
摘要：
背景：浅全基因组测序（Shallow-seq）用于确定组织样品和循环肿瘤DNA中的拷贝数畸变（CNA）。然而,NGS的成本和小型活检的挑战要求替代非靶向NGS方法。mFAST-SeqS方法，依靠LINE-1重复扩增，与Shallow-seq检测血样中的CNA具有良好的相关性。在本研究中,我们评估了mFAST-SeqS是否适合评估小福尔马林固定石蜡包埋（FFPE）组织标本中的CNA，使用外阴和肛门HPV相关病变。
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。
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。
