Keratin 20 is a specific marker of submicroscopic lymph node metastases in colorectal cancer: validation by K‐RAS mutations

Lymph node status has major prognostic importance in colorectal cancer and greater precision in the diagnosis of lymph node metastases should provide better prognostic and therapeutic guidance. Keratin 20 (K20) gene expression has been used as a marker of lymph node metastases, but the evidence for this remains circumstantial. This study has therefore sought to determine K20 specificity and to correlate K20 expression with mutant K‐RAS expression, in order to provide direct evidence that K20 expression in lymph nodes of colorectal cancer patients genuinely reflects metastatic disease. Specificity of K20 expression was established against a range of tissue types and 289 lymph nodes from 41 non‐cancer control patients. K20 expression was restricted to gastrointestinal epithelia and was only present in one of the 289 control lymph nodes, giving a calculated specificity of 97.6% (95% confidence limits: 87.1–99.9%). Forty‐two tumour samples were analysed for the presence of K‐RAS codon 12 gene mutations using a RT‐PCR mutant allele‐specific amplification (MASA) technique. Thirteen tumours (31%) had codon 12 mutations detected by MASA and these were further analysed to determine the exact nature of the mutation. MASA was then used to screen the lymph nodes from these patients for the presence of the tumour‐specific K‐RAS transcript and the results were compared with K20 RT‐PCR and histopathology from the same samples. Whilst K‐RAS MASA was not as sensitive as K20 RT‐PCR, there was substantial agreement between the assays. There were no K20‐negative lymph nodes which were found to be K‐RAS MASA‐positive, whereas seven nodes in four patients were K20‐positive and K‐RAS‐negative, in keeping with the differences in assay sensitivity. These results further validate K20 as a marker by providing greater certainty that what is being detected represents occult metastatic disease. Copyright © 2000 John Wiley & Sons, Ltd.

[1]  J. McCall,et al.  Analysis of potential markers for detection of submicroscopic lymph node metastases in breast cancer , 1999, British Journal of Cancer.

[2]  J. McCall,et al.  Detection and significance of minimal residual disease in colorectal cancer. , 1999, Histology and histopathology.

[3]  J. McCall,et al.  Detection of carcinoembryonic antigen messenger RNA in lymph nodes from patients with colorectal cancer. , 1998, The New England journal of medicine.

[4]  Y. Takagi,et al.  Spread of colorectal cancer micrometastases in regional lymph nodes by reverse transcriptase‐polymerase chain reactions for carcinoembryonic antigen and cytokeratin 20 , 1998, Journal of surgical oncology.

[5]  J. McCall,et al.  Keratin 19 mRNA is detectable by RT-PCR in lymph nodes of patients without breast cancer. , 1997, British Journal of Cancer.

[6]  J. McCall,et al.  Detection of micrometastases in colorectal cancer patients by K19 and K20 reverse-transcription polymerase chain reaction. , 1996, Laboratory investigation; a journal of technical methods and pathology.

[7]  J. Arends,et al.  A detailed analysis of K‐ras point mutations in relation to tumor progression and survival in colorectal cancer patients , 1996, International journal of cancer.

[8]  B. Iacopetta,et al.  Clonal Analysis of Colorectal Tumors Using K-ras and p53 Gene Mutations as Markers , 1995, Diagnostic molecular pathology : the American journal of surgical pathology, part B.

[9]  F. Sarkar,et al.  A Universal Method for the Mutational Analysis of K-ras and p53 Gene in Non‐small-cell Lung Cancer Using Formalin‐Fixed Paraffin‐Embedded Tissue , 1995, Diagnostic molecular pathology : the American journal of surgical pathology, part B.

[10]  K. Müller,et al.  Low specificity of cytokeratin 19 reverse transcriptase-polymerase chain reaction analyses for detection of hematogenous lung cancer dissemination. , 1995, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[11]  R. Fleischmann,et al.  Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. , 1995, Nature.

[12]  M. Ogawa,et al.  Genetic diagnosis of lymph-node metastasis in colorectal cancer , 1995, The Lancet.

[13]  K. Pittman,et al.  Detection of epithelial cancer cells in peripheral blood by reverse transcriptase-polymerase chain reaction. , 1995, British Journal of Cancer.

[14]  E. Kaplan,et al.  K-ras mutations in putative preneoplastic lesions in human colon. , 1993, Journal of the National Cancer Institute.

[15]  J. Bernaudin,et al.  Detection of c-Ki-ras mutation by PCR/RFLP analysis and diagnosis of pancreatic adenocarcinomas. , 1993, Journal of the National Cancer Institute.

[16]  K. Kinzler,et al.  Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. , 1992, Science.

[17]  J. R. Landis,et al.  The measurement of observer agreement for categorical data. , 1977, Biometrics.

[18]  Jacob Cohen.  A Coefficient of Agreement for Nominal Scales , 1960 .

[19]  S. Bustin,et al.  Genetic detection of lymph node micrometastases in patients with colorectal cancer , 1998, The British journal of surgery.

[20]  Adjuvant therapy for patients with colon and rectum cancer. , 1990, Consensus statement. National Institutes of Health Consensus Development Conference.

[21]  R J Salmon,et al.  [Prognostic factors of colorectal cancer]. , 1989, Pathologie-biologie.