Limitations in Molecular Detection of Lymph Node Micrometastasis From Colorectal Cancer

Abstract

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.

RAS Mutations in AUS/FLUS Cytology

“…Fourth, 1 of 5 cytopathologists was involved in cytology interpretation, in which interobserver variability may have existed. Fifth, direct sequencing has been used for mutation analysis in this study, a method which has been known to exclude mutations present in minor fractions of tumor cells, 44 which may have affected our results.…”

KRAS and BRAF mutation analysis can be reliably performed on aspirated cytological specimens of metastatic colorectal carcinoma

“…In this small series, the concordance rate between the paired cytology and histology sample types was 87.5%. Cases of discordant CRC genotype in primary versus metastatic tumour have been reported in the literature in both nodal as well as solid organ metastases relative to the genotype of the primary tumour 14,15,19 . Possible explanations for this phenomenon, aside from laboratory‐related errors, could be due to a genuine heterogeneity in KRAS genotype within sub‐clones of the primary tumour.…”

“…DNA was isolated from 5‐μm thick sections of the formalin‐fixed, paraffin‐embedded resections or cell blocks, or from one entire Diff Quik (DQ)‐ or Papanicolaou (Pap)‐stained smear (for cases 1 and 7) with the QIAamp DNA Micro kit method by Qiagen (Dusseldorf, DE, Germany), according to the manufacturer’s protocol and quantitated by a spectrophotometer. The PCR amplification for the clinically relevant KRAS exons 2 and 3 and BRAF exon 15 were carried out according to published protocols 17,18,19 . The number of sections used and the quantity and quality of DNA extracted before PCR are tabulated in Table 1.…”

“…Moreover, attention has been drawn to the fact that, in a minority of cases, the KRAS status of the primary tumour and the regional nodal or distant metastasis may be different 14,15,19 . Such findings may signal a paradigm shift in the established practice of sequencing the primary tumour for targeted anti‐EGFR receptor therapy in the setting of unresectable metastatic disease.…”

Sequencing of cancer cell subpopulations identifies micrometastases in a bladder cancer patient

“…In the future, perhaps sorting for the more abundant EPCAM + population may improve sensitivity for cancer detection. While previous studies in other cancers have identified micrometastases using RT-PCR, direct Sanger sequencing of specific genes, or histology, our study is the first, to our knowledge, in which whole-exome sequencing has been utilized to prospectively detect and quantify micrometastases in cancer [ 4 , 12 – 14 ]. Furthermore, while intratumoral heterogeneity has previously been studied in urothelial bladder cancer on a chromosomal level, our analysis is the first to examine this using next-generation sequencing [ 15 ].…”