Limitations of cytokeratin 20 RT-PCR to detect disseminated tumour cells in blood and bone marrow of patients with colorectal cancer: expression in controls and downregulation in tumour tissue

Vlems, F. A.; Diepstra, J. Heleen S.; Cornelissen, Ine M. H. A.; Ruers, Theo J.M.; Ligtenberg, M. J. L.; Punt, Cornelis J.A.; Krieken, J.H.J.M. van; Wobbes, Th.; Muijen, G.N.P. van

doi:10.1136/mp.55.3.156

Cited by 61 publications

(39 citation statements)

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“…CTC detection methods also need to be taken into account, as sensitivity and specificity are of major importance and may differ significantly. Thus, it will be important to define the critical variables in the methods and to introduce at least some level of standardization to allow for more reliable and reproducible results (Pantel et al, 1999;Vlems et al, 2002;Uen et al, 2006;Khair et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of MUC1, CK20, and hTERT expression in peripheral blood of gastrointestinal cancer patients in search of diagnostic criteria

Küçükyıldırım¹,

Erdem²,

Saglar³

et al. 2014

Turk J Biol

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The goal of this study was to identify the optimal marker or marker combinations for detection of gastrointestinal malignancies using reverse-transcriptase polymerase chain (RT-PCR) reaction. To detect the presence of tumors, we analyzed mucin 1 (MUC1), cytokeratin 19 (CK19), cytokeratin 20 (CK20), and human telomerase reverse transcriptase (hTERT) mRNA in the peripheral blood of 31 patients with gastrointestinal (esophagus, stomach, and colorectal) cancer and 30 healthy individuals. In RT-PCR analysis of the peripheral blood, 77.4% (24/31), 61.29% (19/31), and 45.16% (14/31) of cancer patients were positive for MUC1, CK20, and hTERT mRNA, respectively. According to our results, any one of these mRNA markers is a predictor of the presence of gastrointestinal tumors (P < 0.001) and colorectal tumors (P < 0.05). However, they did not have predictive potential for presence of metastasis in gastrointestinal tumors. As a result, combination of these 3 tumor-specific mRNA markers would increase the detection rate and may be clinically helpful in predicting tumor presence.

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Section: Discussionmentioning

confidence: 99%

Evaluation of MUC1, CK20, and hTERT expression in peripheral blood of gastrointestinal cancer patients in search of diagnostic criteria

Küçükyıldırım¹,

Erdem²,

Saglar³

et al. 2014

Turk J Biol

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“…A venous catheter was inserted into the right gastric omental veins of gastric cancer patients and corresponding veins of colorectal cancer patients and blood samples were collected. The initial 5 mL blood was discarded to reduce possible contamination and the following 5 mL of blood drawn using a new syringe, was used for RNA extraction [7] . Tissue samples from 27 gastric cancer patients and www.wjgnet.com 19 colorectal cancer patients were formalin-fixed and parafin-embedded.…”

Section: Blood and Tissue Samplesmentioning

confidence: 99%

Relationship between vascular invasion and microvessel density and micrometastasis

Wang¹

2007

WJG

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AIM:To evaluate the relationship between vascular invasion and microvessel density (MVD) of tissue and micrometastasis in blood. M E T H O D S :V a s c u l a r i n v a s i o n w a s d e t e c t e d b y b o t h h e m a t o x y l i n a n d e o s i n s t a i n i n g a n d immunohistochemiscal staining. Blood samples were collected from 17 patients with vascular invasion and 29 patients without vascular invasion and examined for cytokeratin20 (CK20) expression by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Microvessel density of tissue samples was also determined by immunohistochemistry using antibodies to CD105.RESULTS: CK20 was detected in 12 of the 17 patients with vascular invasion and in 9 of the 29 patients without vascular invasion. Positive RT-PCR was significantly correlated with vascular invasion (70.6% vs 30.0%, P < 0.05). The average MVD was significantly higher in patients with positive vascular invasion than in patients with negative vascular invasion (29.2 ± 3.3 vs 25.4 ± 4.7, P < 0.05). The vascular invasion detected with hematoxylin-eosin staining was less than that with immunohistochemical staining. There was a significant difference between the two staining methods (19.6% vs 36.9%, P < 0.05). CONCLUSION:Positive CK20 RT-PCR, depth of tumor invasion, lymph node status, metastasis and MVD are significantly correlated with vascular invasion. Immunohistochemical staining is more sensitive than hematoxylin-eosin staining for detecting vascular invasion.

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Giribaldi

Procida

Ulliers

et al. 2006

The Journal of Molecular Diagnostics

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A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of cytokeratin 20-positive cells in blood characterized by two novel features was developed and tested on 99 patients with colorectal cancer, 110 with breast cancer, and 150 healthy subjects. To optimize the specificity and sensitivity of the method, two novel features were used. First, a primer overlapping two adjacent exons was generated to inhibit nonspecific amplification both in healthy donors and cancer patients; second, a non-end-point first-round amplification was used to increase sensitivity. The number of firstround cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in singleround RT-PCR to 99.0% in nested real-time RT-PCR. To establish sensitivity and specificity of the method, HT29 cells were serially diluted with normal blood. Detection limit improved from 100 HT29 cells (singleround RT-PCR) to 1 to 10 cells (nested real-time RT-PCR) per 3 ml of whole blood. None of the healthy subjects was positive, whereas 22 and 29% of all colorectal and breast cancer patients, respectively, had cytokeratin 20 cell equivalents in blood. The association between cytokeratin 20 cell equivalents and metastasis was statistically significant for breast (P ‫؍‬ 0.026) but not colorectal cancer patients (P ‫؍‬ 0.361). Negativity of all 150 healthy controls examined confers diagnostic potential to the method. Cytokeratin mRNAs are potential markers for detection of epithelial cells in blood. Several reports indicate that cytokeratin 20 (CK20) mRNA in blood acts as a specific cancer cell marker in patients with frequent cancer forms of epithelial origin such as breast 1-5 and colorectal cancer.6 -9 Many breast cancer patients develop metastasis after locoregional and systemic treatment even in the absence of dissemination as assessed by conventional diagnostic tools. Approximately 30 to 50% of colorectal cancer patients who have undergone curative resection have recurrences with fatal outcome.10,11 Most recurrences occur in patients with TNM (tumor, nodes, and metastases) stage II and III cancers, but patients with stage I lesions also have appreciable risk. In these patients, cancer cells were disseminated either before or during surgery of the primary tumor.12-14 Although the relationship between circulating tumor cells and the development of recurrent cancer is not fully understood, it is generally assumed that enhanced dissemination of cancer cells in blood contributes significantly to the development of metastasis. [15][16][17] The detection of circulating metastatic cells would be of great value for the assessment of the metastatic risk.18 Currently, the most powerful prognostic information in cancer patients is obtained from conventional histological assessment of regional lymph nodes. 19 -23 Because 20 to 30% of colorectal cancer patients without metastasis in lymph nodes die from distant metastases or local recurrence within 5 years 24...

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Limitations of cytokeratin 20 RT-PCR to detect disseminated tumour cells in blood and bone marrow of patients with colorectal cancer: expression in controls and downregulation in tumour tissue

Cited by 61 publications

References 38 publications

Evaluation of MUC1, CK20, and hTERT expression in peripheral blood of gastrointestinal cancer patients in search of diagnostic criteria

Evaluation of MUC1, CK20, and hTERT expression in peripheral blood of gastrointestinal cancer patients in search of diagnostic criteria

Relationship between vascular invasion and microvessel density and micrometastasis

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

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