Transcription of Cytokeratins 8, 18, and 19 in Bone Marrow and Limited Expression of Cytokeratins 7 and 20 by Carcinoma Cells: Inherent Limitations for RT-PCR in the Detection of Isolated Tumor Cells

Abstract

SUMMARY:The suitability of "real-time" quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of isolated carcinoma cells in bone marrow was investigated by evaluating the expression of cytokeratin (CK)7, CK8, CK18, CK19, and CK20 in 17 gastrointestinal cancer cell lines, 64 control bone marrow specimens from noncancer patients, and 30 bone marrow specimens from patients with gastric or colorectal cancer. RT-PCR products for CK8 and CK18 were detected in all cancer cell lines, but only 16, 5, and 11 cell lines provided evidence for CK19, CK7, and CK20 transcription. Variable numbers of bone marrow specimens from noncancer patients demonstrated background transcription of CK8 (78.1%), CK18 (95.3%), CK19 (35.9%), CK20 (29.6%), and CK7 (16.7%). Maximal background transcription for CK8, CK18, and CK19 ranged from 52.2 to 56.1 copies/10 3 copies glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the corresponding values of 0.06 and 0.76 copies for CK7 and CK20 being distinctly lower. When maximal background values were used as a threshold value to define positivity in tumor cell dilution experiments, sensitivity levels of one tumor cell in 10 4 bone marrow cells were determined for CK7 and CK20 RT-PCR assays. Maximal background expression values of the different CKs as obtained in the control series were exceeded once (CK20), twice (CK18 and CK19), and 18 times (CK7) in bone marrow specimens from cancer patients, with none of these specimens exceeding the maximal background expression value of CK8. We conclude that RT-PCR for CK8, CK18, and CK19 cannot be recommended for the detection of isolated tumor cells in bone marrow of cancer patients. On the other side, the limited number of gastric and colorectal cancer cell lines expressing CK7 and CK20 indicates that assay sensitivity for these CKs might be limited because of their selective expression by carcinoma cells. (Lab Invest 2001, 81:1351-1361.

Real‐time RT‐PCR detection of CK19, CK7 and MUC1 mRNA for diagnosis of lymph node micrometastases in non small cell lung carcinoma

“…39 Transcription of CK7 has been evaluated by Dimmler et al 40 using Taqman chemistry for detection of carcinoma cells mixed with bone marrow cells, showing a sensitivity level of 1 tumor cell per 10 4 bone marrow cells. In our study, the sensitivity of detection using CK7 mRNA in serial dilutions of A549 was identical to that reported by Dimmler et al, 40 i.e., 1 tumor cell in 10 4 PBMNs.…”

“…39 Transcription of CK7 has been evaluated by Dimmler et al 40 using Taqman chemistry for detection of carcinoma cells mixed with bone marrow cells, showing a sensitivity level of 1 tumor cell per 10 4 bone marrow cells. In our study, the sensitivity of detection using CK7 mRNA in serial dilutions of A549 was identical to that reported by Dimmler et al, 40 i.e., 1 tumor cell in 10 4 PBMNs. The sensitivity of CK19 and CK7 mRNAs detection by realtime RT-PCR evaluated by serial dilutions of standards is similar to the results reported by D'Cunha et al, 41 who tested the accuracy of ACE mRNA quantification using real-time RT-PCR with Taqman chemistry for the detection of LN micrometastases.…”

Detection of disseminated pancreatic cells by amplification of cytokeratin-19 with quantitative RT-PCR in blood, bone marrow and peritoneal lavage of pancreatic carcinoma patients

“…Reverse transcriptase polymerase chain reaction (RT-PCR) has a high sensitivity and allows the identification of approximately 1 tumor cell in 10 7 normal peripheral mononuclear blood cells [13] . Using qualitative PCR methods, varying frequency of gene transcripts and false positive results have been reported [14][15][16] . Therefore, tumor cell detection in patients with ductal adenocarcinoma of the pancreas is still a matter of debate.…”

Specific Detection of Cytokeratin 20-Positive Cells in Blood of Colorectal and Breast Cancer Patients by a High Sensitivity Real-Time Reverse Transcriptase-Polymerase Chain Reaction Method

“…CK20 is considered a marker of neoplastic epithelial cells, but circulating CK20 ϩ cells were described to be present or absent in the blood of both cancer patients and healthy subjects ( 7,(32)(33)(34)(35)38 ). These apparently conflicting results were not surprising because we (G. Giribaldi, unpublished data) and others 39 frequently obtained false-positive results by using conventional nested RT-PCR methods.…”

“…Moreover, current methods are often not quantitative, 1,31 have low sensitivity, 6,31 possess high risk of cross-contamination, and have low specificity. 2,7,[32][33][34][35] Very few reports compared results obtained in different cancer types by the same methodology 36 to ascertain whether differences were due to the cancer type or to the method used.…”