Klebig Felix, Fischer Carsten, Petri Susan, Gerull Helwe, Wagener Christoph, Tschentscher Peter
Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20251 Hamburg, Germany.
Diagn Mol Pathol. 2007 Jun;16(2):91-5. doi: 10.1097/PDM.0b013e31803278ee.
Colorectal cancer patients with lymph node metastasis have a shorter survival and may require adjuvant therapy after surgery of the primary tumor. It is supposed that a more reliable diagnosis can be achieved using tumor-specific DNA mutations for the detection of metastasizing cells. To design a practical approach for a molecular diagnosis of micrometastasis, we applied direct DNA sequencing to screen 48 early stage colorectal carcinomas for the most frequent mutations of the KRAS, P53, and APC tumor genes. KRAS mutations were detected as frequently as described earlier. In contrast, the frequency of P53 and APC hot spot mutations was unexpectedly low, compared with previous studies using other screening methods or including advanced tumor stages. Not more than 31% of early stage tumors showed a mutation in at least 1 of the selected hot spot codons. Applying mutant-enriched polymerase chain reaction (PCR), the mutation of the primary tumor was detected in lymph node DNA from 2 of the KRAS-positive patients. In 1 patient, the result was not verified by subtractive iterative PCR, a principally different molecular method with high sensitivity and specificity. Our data suggest that screening for suitable markers for a molecular detection of occult lymph node metastasis cannot be restricted to small-sized hot spot regions of a few tumor genes and possibly must include tumor-specific epigenetic changes. Furthermore, restriction enzyme-based methods such as mutant-enriched PCR are not suitable to detect any mutation with equal efficiency and they should be carefully controlled to avoid false-positive detection of marker mutations in lymph node DNA.
有淋巴结转移的结直肠癌患者生存期较短，原发肿瘤手术后可能需要辅助治疗。据推测，利用肿瘤特异性DNA突变检测转移细胞可实现更可靠的诊断。为设计一种实用的微转移分子诊断方法，我们应用直接DNA测序技术对48例早期结直肠癌进行筛查，以检测KRAS、P53和APC肿瘤基因最常见的突变。KRAS突变的检测频率与先前报道的一致。相比之下，与使用其他筛查方法或纳入晚期肿瘤阶段的先前研究相比，P53和APC热点突变的频率意外较低。不超过31%的早期肿瘤在至少1个选定的热点密码子中出现突变。应用突变富集聚合酶链反应（PCR），在2例KRAS阳性患者的淋巴结DNA中检测到原发肿瘤的突变。在1例患者中，该结果未通过消减迭代PCR验证，消减迭代PCR是一种具有高灵敏度和特异性的截然不同的分子方法。我们的数据表明，筛选用于隐匿性淋巴结转移分子检测的合适标志物不能局限于少数肿瘤基因的小尺寸热点区域，可能还必须包括肿瘤特异性表观遗传变化。此外，基于限制性酶的方法如突变富集PCR并不适合以同等效率检测任何突变，应谨慎控制以避免在淋巴结DNA中出现标志物突变的假阳性检测。
