The Investigation Of Measuring CK18 Expression By Real Time Fluorescence Quantitative PCR For Diagnosis Of Lymph Node Micrometastasis In Gastric Carcinomas

The final diagnosis of lymph nodes( LNs)micrometastasis in cancer comes from the detection by immunohistological techniques, whereas missing diagnosis probably occurs some times. Examination of consecutive sections is usually needed for finding the micrometastatic lesions less than 0.2 mm, which means that there are less than 20 cancerous cells in a single LN. The methods of real time fluorescence quantitative PCR possess benefits of fine specificity, rare rate of fasle positive, and lowerer possibility for missed diagnosis as the intact nodes are used as the source of templates. Therefore, it avoids laborous protocals of consecutive sections in immunopathological examination and discoveries the micrometastasis at a single cell level with specificity and reliability. The key for applying the method is to choose a specific marker for the detection. Cytokeratins (CKs) are a kind of protein of intermediate filaments, with molecular weights of 40 to 68 kDa. CKs are commonly recognized as the tailor-made component of epithelial cells. They exist in heterodimers and are polymerized to form the cytoskeleton with organ or tissue specificities. CK18 are reported as such a marker for glandular epithelium and rich in digestive system. In normal situation, there is little or no expression of CK18 in peripheral blood, marrow and lymph nodes. It is why we select CK18 as the genetic flagging to identify cancerous matestasis in gastric carcinomas. On the basis of investigation for the correlation between micrometastasis of gastric carcinomas and the RT-PCR detection with cytokeratin 18 (CK18) expression as hallmarker, we develop an objective and highly sensitive assay system of RTFQ-PCR to determine the copy numbers of CK18 mRNA in LNs, whereby to identify micrometastasis of epithelial carcinoma cells.A recombinant plasmid with CK18 encoding sequence was established under identification by agarose electrophoresis after PCR, restrict enzyme cleavage and sequencing. RTFQ-PCR of the recombinant plasmid as templates showed that a well linear relationship was present between Ct values and logarithm of the target copies with little of either the inner error or the inter error (CV less than 5% for the standard curve), and sensitivity reached as few as 10 copies of the target fragments. It was found much more superior than ordinary semiquantative PCR when compared in the detection with serial dilutions of the recombinant plasmid. As the result, we ensured that the RTFQ-PCR assay was suitable for detection of minor metastatic lesions clinically .In quantificaion of CK18 mRNA in lymph nodes (LNs) of gastric carcinomas by the RTFQ-PCR assay, We found that no positive expression of CK18 mRNA in the LNs removed from benign gastric ulcer and also, no negative for CK18 mRNA in LNs of both pathologically diagnosed metastasis and the tissues of gastric carcinomas. Therefore, measuring CK18 mRNA in LNs be a reliable hallmarker for diagnozing metastasis of gastric cacinomas. In addition, it might imply that the the measurements reflected occurrence of the metastatic cancer cells and their growth situation in LNs.Micrometastasis of cancerous cells may lead to recurrence of carcinoma, whereas there seems a contradiction for the correlation of LN micrometastasis with prognosis of cancer. In the investigation, a great diversity was found in copy numbers of CK18 mRNA in the LNs of the 66 patients with gastric carcinomas, ranging from zero to more than 108. It was apparent that the amount of metastatic tumor cells was in different levels in the patients. Based on the observation, we analyzed the determination data with combination to TNM staging for the enrolled cases. 6 patients were diagnozed as TNM1, with their copies of CK18mRNA from 0 to 4.28×104. 15 were in TNM2. CK18 mRNA 0 copy was in 5 of them, 8 cases were in 104 magnitude and only two were 7.1×105. On the other hand, 45 patients were grouped in etherⅢo rⅣstage by pathology and CK18 mRNA in LNs less than 104 only was seen in 4 cases, 6 cases's CK18mRNA expression level was 104-105, with 35 ones beyond 105. Therefore, it is concluded that measurement of CK18 mRNA provided some important information helpful to clinicians to judge how severe it is in gastric carcinomas.According to the newest staging standard in AJCC Cancer Staging manual compiled by American Joint Committee on Cancer, 24 cases who were diagnosed N0 pathologically were upgraded to N1 (11 cases) and N2 (12 cases) by the gene diagnosis of RTFQ-PCR. In the 42 cases who were diagnosed N1-N3 pathologically, all were positive by gene diagnosis. 8 cases were changed in staging: a patient was upgraded fromⅠtoⅢA, another fromⅡA toⅢA, and 6 fromⅡB toⅢA. The detection of micrometastasis made 8 cases renewed their staging, with the rate of renewing 33.33%(8/24cases). So, we should do routine detection of LNs by RTFQ-PCR in staging PT3N0. It made TNM staging of gastric carcinomas extended to molecular level, which might reflect real status of metastasis, and be helpful for guiding clinical therapy, monitoring tumor prognosis and recurrence.The expression of CK18 was not totally negative by RTFQ-PCR in the LNs of metastasis-negative diagnosed pathologically. This showed that applying the more sentitive method of gene diagnosis may upgrade the detection rates of micrometastasis in gastric carcinomas. 62.50% of patients who were considered negative metastasis in LNs by routine pathological diagnosis were found micrometastasis in LNs. The copies of CK18mRNA in nonmetastssis was 0, the expression level of CK18 mRNA in micrometastsis and metastasis increased gradually. But the expressing level of CK18mRNA in the 15 cases with micrometastasis was varied significantly. According to corresponding relationship between CK18 mRNA copies in LNs and UICC grading standard for micrometastasis, 2 cases whose CK18 mRNA copies were less than 104, indicating existence of dormacy cells in their LNs. In 5 cases, CK18 mRNA copies in LNs reached 104 to 106, with meaning for the submicrometastasis of 1 to 20 cancer cells per LN. In 8 cases, CK18 mRNA copies were beyond 106, which meant active micrometastasis with 20 to 200 cancer cells per LN. Therefore, the real time quantitation for CK18mRNA expression had rather considerable significance, reflecting the number of metastatic cancer cells in LN directly and precisely.The expressing level of CK18mRNA had close correlation wih size of tumor, depth of tissue infiltration, degree of differentiation, metastasis of LNs and TNM staging. It was indeed true no widely accepted common-norm for the relationship between quantifying the number of epithelial transcripts in LNs and tumor micrometastasis as yet, so that it was unable to reveal, practically, clinical significance of quantifying CK18 transcripts in LNs until now. We define the measured CK18 transcripts beyond 104 of magnitude as the occurrence of the active micrometastasis (not the isolated tumor cells, ITCs), abiding by recent achievements in micrometastasis as well as the definition for high-abundance of a given mRNA species in a single cell. According to the criterion, 52 cases in the 66 patients be considered as ones with active metastasis.Conclusion: the technique of RTFQ-PCR for CK18 mRNA in LNs may detect accurately the micrometastasis of gastric carcinomas.