Molecular detection of lymph node micrometastases in colorectal cancer patients
Authors: Caroline Wood, Hartley Stern, Maha Guindi, Alain E. Lagarde
Journal: Clinical Cancer Research (2006)

A53   Objective: The clinical significance of lymph node micrometastases detected by immunological and molecular methods in node-negative colorectal cancer patients remains to be confirmed. In this study we compared the sensitivity of detection methods based on the transcriptional expression of several epithelial cell-specific genes in paraffin-embedded mesenteric lymph nodes, and assessed survival outcomes. Methods. Each block contained a variable number of nodes. RNA was extracted from 2 serial sections (20 μm each) and submitted to reverse transcription (RT). One seventh of cDNA was PCR amplified for 40 cycles at 60C, using gene-specific primers. Products were resolved by polyacrylamide gel electrophoresis with a DNA mass ladder for quantitation. RNA integrity was verified by amplifying the GAPDH and selenoprotein P housekeeping genes.To estimate the relative number of metastatic cells present in lymph nodes a calibration curve was established by serial dilutions of total RNA from HCT116 cells (10 picog per cell equivalent) followed by RT-PCR. The lowest detection limits were 2, 15-30 and 60 cells for CEA, EZH2 and PRL3 respectively.The study group included 44 patients, 98 blocks and 437 lymph nodes;144 from 18 stage I patients; 124 from15 stage II; 119 from 7 stage III and 50 from 4 stage IV patients. On average 10 nodes were dissected per patient. Fifteen nodes (12.5%) of stage III cases and 7 nodes (14%) of stage IV were identified by hitopathology. Results. Several genes, always expressed in lymph nodes, proved unsuitable markers of micrometastases:cytokeratins (CK20), the CDX2 homeobox gene, osteopontin, decorin. MUC2 was weakly expressed in the nodes of 6 patients (14%). Carcinoembryonic antigen (CEA), enhancer of zeste (EZH2), and the tyrosine phosphatase PRL3 were differentially expressed. EZH2 was always co-expressed with either CEA or PRL3. EZH2-expressing cells were found in 6% of stage I patients but in >30% of stage II, III and IV patients. The proportion of cases with an estimated >50 PRL3-expressing cells per 10,000 node cells increased from 44% (8/18) for stage I, to 87% (13/15, stage II), 86% (6/7, stage III) and 75% (3/4, stage IV). Likewise, the proportion of cases with an estimated >10 CEA-expressing cells per 10,000 node cells increased from 22% in stage I, to 60% (stage II), to 71% (stage III). The proportion of cases in which CEA and PRL3 were highly co-expressed in lymph nodes also increased from 22% (stage I) to >70% in advanced stages.Three of 7 stage III (43%) and 3 of 4 stage IV patients (75%) succumbed to distant metastases within less than 5 years after surgery. All had received chemotherapy. In all 6 cases, high proportions of either CEA-(3 cases) or PRL3-expressing cells (5 cases) or both (3 cases) were found in their lymph nodes. The ability of the CEA and EZH2 assays to predict the outcome of node-negative patients is based on the findings that:  (i)  The single stage I patient who succumbed to the disease after 5 years demonstrated undetectable CEA and PRL3 cells in lymph nodes.The median follow up time of 12 other stage I patients, who expressed CEA alone (6), PRL3 alone (2), both CEA and PRL3 (2) and no marker was 55 months.  (ii)  Five of 15 stage II patients (33%) died between 11 months and 6 years after surgery, 2 of unrelated cancers.They all expressed either CEA, PRL3 or both (3cases) in lymph nodes. Eleven of 15 stage II patients (73%) highly co-expressed CEA and PRL3 in lymph nodes. Three of 11 (27%) succumbed to the disease. Conclusions. We conclude that CEA, PRL3 and EZH2-expressing cells are detectable in lymph nodes in amounts and frequencies consistent with the staged progression of the disease. A higher proportion of stage II-III than stage I cases co-express CEA and PRL3 in lymph nodes.High expression of either CEA, PRL3 or both may account for about 30% of stage II patients with poor prognosis and is not predictive of poor outcomes for stage I patients.