KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment
KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment
复制标题
DOI：
10.1038/modpathol.2011.210
复制
发表时间：
2012-05-01
期刊：
影响因子：
7.5
通讯作者：
Bibeau, Frederic
中科院分区：
文献类型：
--
作者：
Boissiere-Michot, Florence;Lopez-Crapez, Evelyne;Bibeau, Frederic
在接受抗表皮生长因子受体单克隆抗体治疗前，对转移性结直肠癌患者进行KRAS状态评估是强制性的，因为KRAS突变与对这种治疗的耐药性有关。然而，KRAS基因分型在肿瘤细胞性差的情况下可能非常具有挑战性，特别是当局部晚期直肠腺癌在放化疗后实现主要肿瘤消退时。我们的目标是确定最可靠的策略来检测这些样本中的KRAS突变。从31例主要肿瘤消退的手术标本中提取DNA，经过手工解剖，并从配对的治疗前活检中提取DNA，并进行高分辨率熔化分析。然后对显示熔化曲线形状改变的DNA样本进行测序。使用等位基因特异性PCR （Thera Screen）和激光显微解剖（随后进行高分辨率熔化和测序分析）进一步研究未修改熔化曲线或野生型序列的样品。在31例放化疗后手术标本中，通过高分辨率熔化分析/测序鉴定出7个KRAS突变。通过Thera筛选试验检测到一个额外的突变，在激光显微解剖后检测到两个突变，包括Thera筛选试验鉴定的突变。总共有9/31例手术标本（29%）出现KRAS突变。在人工解剖的治疗前活检中，通过高分辨率熔化分析和测序鉴定出12个突变（39%）。使用Thera筛选试验或激光显微解剖未发现其他突变。这些结果表明，对于低肿瘤细胞的结直肠癌放化疗后手术标本，治疗前活检可能是可靠的KRAS基因分型的最具成本效益的选择。可以设想使用更灵敏的检测方法，如等位基因特异性PCR或激光显微解剖，但成本更高，延误时间更长。现代病理学杂志（2012）25,731-739；doi: 10.1038 / modpathol.2011.210;2012年1月27日在线发布
KRAS status assessment is mandatory in patients with metastatic colorectal cancer before therapy with anti-epidermal growth factor receptor monoclonal antibodies, as KRAS mutations are associated with resistance to this treatment. However, KRAS genotyping may be very challenging in case of poor tumor cellularity, particularly when major tumor regression is achieved in locally advanced rectal adenocarcinomas after radiochemotherapy. We aimed at identifying the most reliable strategy to detect KRAS mutations in such samples. DNA was extracted from 31 surgical specimens with major tumor regression, following manual dissection, and from paired pre-treatment biopsies and analyzed by high-resolution melting. DNA samples displaying altered melting curve shapes were then sequenced. Samples with unmodified melting curves or wild-type sequence were further investigated by using an allele-specific PCR assay (Thera Screen) and laser microdissection (followed by high-resolution melting and sequencing analyses). In the 31 post-radiochemotherapy surgical specimens, seven KRAS mutations were identified by high-resolution melting analysis/sequencing. One additional mutation was detected by the Thera Screen assay and two mutations, including the one identified by the Thera Screen assay, were detected following laser microdissection. Altogether, 9/31 surgical specimens (29%) presented KRAS mutations. In the manually dissected pre-treatment biopsies, 12 mutations (39%) were identified by high-resolution melting analysis and sequencing. No additional mutations were found by using the Thera Screen assay or laser microdissection. These results indicate that, in the case of post-radiochemotherapy surgical specimens of colorectal cancer with low tumor cellularity, pre-treatment biopsies might represent the most cost-effective option for reliable KRAS genotyping. The use of more sensitive assays, such as allele-specific PCR or laser microdissection, can be envisaged but with higher costs and longer delays. Modern Pathology (2012) 25, 731-739; doi:10.1038/modpathol.2011.210; published online 27 January 2012