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van der Toom EE, Axelrod HD, de la Rosette JJ, de Reijke TM, Pienta KJ, Valkenburg KC. Prostate-specific markers to identify rare prostate cancer cells in liquid biopsies. Nat Rev Urol 2019; 16:7-22. [PMID: 30479377 DOI: 10.1038/s41585-018-0119-5] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Despite improvements in early detection and advances in treatment, patients with prostate cancer continue to die from their disease. Minimal residual disease after primary definitive treatment can lead to relapse and distant metastases, and increasing evidence suggests that circulating tumour cells (CTCs) and bone marrow-derived disseminated tumour cells (BM-DTCs) can offer clinically relevant biological insights into prostate cancer dissemination and metastasis. Using epithelial markers to accurately detect CTCs and BM-DTCs is associated with difficulties, and prostate-specific markers are needed for the detection of these cells using rare cell assays. Putative prostate-specific markers have been identified, and an optimized strategy for staining rare cancer cells from liquid biopsies using these markers is required. The ideal prostate-specific marker will be expressed on every CTC or BM-DTC throughout disease progression (giving high sensitivity) and will not be expressed on non-prostate-cancer cells in the sample (giving high specificity). Some markers might not be specific enough to the prostate to be used as individual markers of prostate cancer cells, whereas others could be truly prostate-specific and would make ideal markers for use in rare cell assays. The goal of future studies is to use sensitive and specific prostate markers to consistently and reliably identify rare cancer cells.
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Affiliation(s)
| | - Haley D Axelrod
- The James Buchanan Brady Urological Institute, Baltimore, MD, USA.,Graduate Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | | | | | - Kenneth J Pienta
- The James Buchanan Brady Urological Institute, Baltimore, MD, USA
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2
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Qiu MZ, Li ZH, Zhou ZW, Li YH, Wang ZQ, Wang FH, Huang P, Aziz F, Wang DY, Xu RH. Detection of carcinoembryonic antigen messenger RNA in blood using quantitative real-time reverse transcriptase-polymerase chain reaction to predict recurrence of gastric adenocarcinoma. J Transl Med 2010; 8:107. [PMID: 21040522 PMCID: PMC2989934 DOI: 10.1186/1479-5876-8-107] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2009] [Accepted: 10/31/2010] [Indexed: 12/27/2022] Open
Abstract
Background The existence of circulating tumor cells (CTCs) in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma. Methods Blood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA) mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging. Results The minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA) × 106] was set at 100. Forty-five of 123 patients (36.6%) were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001). Recurrent disease was found in 44 of 123 cases (35.8%), and 25 of these (56.8%) were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P < 0.001). Only histological grade and CEA mRNA positivity were independent factors for disease-free survival using multivariate analysis. Conclusions CEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients.
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Affiliation(s)
- Miao-Zhen Qiu
- State Key Laboratory of Oncology in South China, Guangzhou 510060, China
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3
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Kowalewska M, Nowak R, Chechlinska M. Implications of cancer-associated systemic inflammation for biomarker studies. Biochim Biophys Acta Rev Cancer 2010; 1806:163-71. [PMID: 20600631 DOI: 10.1016/j.bbcan.2010.06.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2010] [Revised: 06/16/2010] [Accepted: 06/17/2010] [Indexed: 12/19/2022]
Abstract
Highly sensitive molecular technologies provide new capacities for cancer biomarker research, but with sensitivity improvements marker specificity is significantly decreased, and too many false-positive results should disqualify the measurement from clinical use. Hence, of the thousands of potential cancer biomarkers only a few have found their way to clinical application. Differentiating false-positive results from true-positive (cancer-specific) results can indeed be difficult, if validation of a marker is performed against inadequate controls. We present examples of accumulating evidence that not only local but also systemic inflammatory reactions are implicated in cancer development and progression and interfere with the molecular image of cancer disease. We analyze several modern strategies of tumor marker discovery, namely, proteomics, metabonomics, studies on circulating tumor cells and circulating free nucleic acids, or their methylation degree, and provide examples of scarce, methodologically correct biomarker studies as opposed to numerous methodologically flawed biomarker studies, that examine cancer patients' samples against those of healthy, inflammation-free persons and present many inflammation-related biomarker alterations in cancer patients as cancer-specific. Inflammation as a cancer-associated condition should always be considered in cancer biomarker studies, and biomarkers should be validated against their expression in inflammatory conditions.
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Affiliation(s)
- Magdalena Kowalewska
- Department of Molecular Biology, Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Roentgena 5, 02-781 Warsaw, Poland
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4
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Chechlinska M, Kowalewska M, Nowak R. Systemic inflammation as a confounding factor in cancer biomarker discovery and validation. Nat Rev Cancer 2010; 10:2-3. [PMID: 20050335 DOI: 10.1038/nrc2782] [Citation(s) in RCA: 123] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Affiliation(s)
- Magdalena Chechlinska
- Department of Immunology, Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Warsaw, Poland.
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5
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Hoffmann K, Kerner C, Wilfert W, Mueller M, Thiery J, Hauss J, Witzigmann H. 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. World J Gastroenterol 2007; 13:257-63. [PMID: 17226905 PMCID: PMC4065954 DOI: 10.3748/wjg.v13.i2.257] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To evaluate the diagnostic potential of cytokeratin-19 (CK-19) mRNA for the detection of disseminated tumor cells in blood, bone marrow and peritoneal lavage in patients with ductal adenocarcinoma of the pancreas.
METHODS: Sixty-eight patients with pancreatic cancer (n = 37), chronic pancreatitis (n = 16), and non-pancreatic benign surgical diseases (n = 15, control group) were included in the study. Venous blood was taken preoperatively, intraoperatively and at postoperative d 1 and 10. Preoperative bone marrow aspirates and peritoneal lavage taken before mobilization of the tumor were analyzed. All samples were evaluated for disseminated tumor cells by CK-19-specific nested-PCR and quantitative fluorogenic RT-PCR.
RESULTS: CK-19 mRNA expression was increased in 24 (64%) blood samples and 11 (30%) of the peritoneal lavage samples in the patients with pancreatic cancer. In 15 (40%) of the patients with pancreatic cancer, disseminated tumor cells were detected in venous blood and bone marrow and/or peritoneal lavage. In the peritoneal lavage, the detection rates were correlated with the tumor size and the tumor differentiation. CK-19 levels were increased in pT3/T4 and moderately/poorly differentiated tumors (G2/G3). Pancreatic cancer patients with at least one CK-19 mRNA-positive sample showed a trend towards shorter survival. Pancreatic cancer patients showed significantly increased detection rates of disseminated tumor cells in blood and peritoneal lavage compared to the controls and the patients with chronic pancreatitis.
CONCLUSION: Disseminated tumor cells can be detected in patients with pancreatic ductal adenocar-cinoma by CK-19 fluorogenic RT-PCR. In peritoneal lavage, detection rate is correlated with tumor stage and differentiation. In the clinical use, CK-19 is suitable for the distinction between malignant and benign pancreatic disease in combination with other tumor-specific markers.
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MESH Headings
- Biomarkers, Tumor/analysis
- Biomarkers, Tumor/blood
- Bone Marrow/pathology
- Carcinoma, Pancreatic Ductal/diagnosis
- Carcinoma, Pancreatic Ductal/pathology
- Humans
- Keratin-19/genetics
- Neoplastic Cells, Circulating/chemistry
- Neoplastic Cells, Circulating/pathology
- Pancreatic Neoplasms/diagnosis
- Pancreatic Neoplasms/pathology
- Peritoneal Lavage
- RNA, Messenger/analysis
- RNA, Messenger/blood
- RNA, Neoplasm/analysis
- RNA, Neoplasm/blood
- Reverse Transcriptase Polymerase Chain Reaction/methods
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Affiliation(s)
- Katrin Hoffmann
- Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University of Leipzig, Germany.
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Giribaldi G, Procida S, Ulliers D, Mannu F, Volpatto R, Mandili G, Fanchini L, Bertetto O, Fronda G, Simula L, Rimini E, Cherchi G, Bonello L, Maule MM, Turrini F. 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. J Mol Diagn 2006; 8:105-12. [PMID: 16436641 PMCID: PMC1867572 DOI: 10.2353/jmoldx.2006.050054] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
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 first-round cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in single-round 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 (single-round 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.
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Affiliation(s)
- Giuliana Giribaldi
- Università di Torino, Dipartimento di Genetica, Biologia e Biochimica, Via Santena 5 bis, 10126 Torino, Italy.
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7
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Suzuki A, Yagisawa J, Kumakura SI, Tsutsui T. Effects of minocycline and doxycycline on cell survival and gene expression in human gingival and periodontal ligament cells. J Periodontal Res 2006; 41:124-31. [PMID: 16499715 DOI: 10.1111/j.1600-0765.2005.00843.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
OBJECTIVES AND BACKGROUND Periodontitis is an infectious disease in the gingival crevice caused by periodontopathic bacteria, including Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, and Tennerella forsythensis, and antibacterial agents are directly administered to the site of infection to treat it. To maximize the therapeutic effects while reducing the adverse effects, the antibacterial agents should be administered at concentrations greater than their MIC(90) doses required to inhibit the growth of 90% of periodontopathic bacteria and the administration should not damage the periodontal tissue. One approach for estimating cellular damage in the periodontal tissue caused by the administration is to assay cytological damages following exposures of cultured human cells derived from periodontal tissues to antibacterial agents. In the present study, we investigated the cytotoxic effect of minocycline (MINO) and doxycycline (DOX) by using a human gingival fibroblast cell line, a human gingival epithelial cell line, and a human periodontal ligament fibroblast cell line. We also used these cell lines to study the effect of MINO or DOX on the mRNA and protein expressions of genes associated with the differentiation of fibroblasts and the proliferation, differentiation, or cellular adhesion important to the epithelial regeneration of the periodontal attachment. METHODS The cytotoxic effect of MINO or DOX was measured as a decrease in cell survivals. The effects of these antibiotics on the mRNA and protein expressions in the cell lines were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively. RESULTS The maximum concentration of MINO or DOX that has little effect on the cell survivals and the mRNA and protein expressions of genes for alkaline phosphatase, type I procollagen, keratinocyte growth factor receptor, keratin 18 or 8/18, integrin beta1, integrin beta4, and laminin 5gamma2 was 10 or 30 microm, respectively, which are greater than their MIC(90) doses against periodontopathic bacteria described above. CONCLUSIONS These findings suggest that little, if any, cellular damage would be expected with topical administration of MINO or DOX to the periodontal pocket at a dose equivalent to the MIC(90). It is important to note, however, that the extrapolation of these findings to in vivo conditions has yet to be undertaken.
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Affiliation(s)
- Ayako Suzuki
- Department of Pharmacology, The Nippon Dental University, School of Dentistry at Tokyo, Japan
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Hsu CP, Shen GH, Ko JL. Matrix metalloproteinase-13 expression is associated with bone marrow microinvolvement and prognosis in non-small cell lung cancer. Lung Cancer 2006; 52:349-57. [PMID: 16569461 DOI: 10.1016/j.lungcan.2006.01.011] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2005] [Revised: 01/10/2006] [Accepted: 01/31/2006] [Indexed: 11/16/2022]
Abstract
Our previous study demonstrated that bone marrow microinvolvement (BMM) is an epiphenomenon of tumor progression rather than a prognostic factor in non-small cell lung cancer. We hypothesize that an increase in mesenchymal transition power in epithelial tumor cells by up-regulation of the matrix metalloproteinases (MMPs) may contribute to the existence of BMM and poorer prognosis. Hereby we conducted a prospective study of BMM and MMPs expression in a cohort of 57 non-small cell lung cancer patients. Bone aspirates were examined by immunohistochemical stains. Expressions of MMPs were checked by Human MMP primer set kit (Maxim Biotech, USA). Correlations between the MMPs expression and BMM, nodal metastasis, and prognosis were examined. Cox model analysis was used to identify independent prognostic factors. Though positive BMM was identified in 38 (66.7%) of the patients, none of the clinicopathological factors, including sex, age, cell types, tumor differentiation, nodal metastasis and TNM status of the tumor, was related to BMM by the tumor cells. Up-regulation was observed in a broad spectrum of MMPs with the exception of MMP-3. However, only MMP-13 expression correlated with the existence of BMM (p=0.006). Univariate analysis revealed MMP-3, MMP-7 and MMP-13 as negative prognostic factors. Cox model analysis revealed T-status, cell differentiation, and MMP-13 expression of the tumor as independent prognostic factors. The overall 5-year survival rate of the patients was 36.8%. The existence of BMM itself did not influence the prognosis (p=0.109), however, patients with positive MMP-13 expression (N=34) had a poorer 5-year survival rate of 26.5% (p=0.025). In summary, non-small cell lung cancer cells with MMP-13 expression, despite of BMM status, tend to shed and aggregate in the bone marrow, which is subsequently reflected in a poorer survival rate.
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Affiliation(s)
- Chung-Ping Hsu
- Division of Thoracic Surgery, Department of Surgery, Taichung Veterans General Hospital, Taichung, and School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC.
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9
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Newton TR, Parsons PG, Lincoln DJ, Cummings MC, Wyld DK, Webb PM, Green AC, Boyle GM. Expression profiling correlates with treatment response in women with advanced serous epithelial ovarian cancer. Int J Cancer 2006; 119:875-83. [PMID: 16557592 DOI: 10.1002/ijc.21823] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The majority of epithelial ovarian carcinomas are of serous subtype, with most women presenting at an advanced stage. Approximately 70% respond to initial chemotherapy but eventually relapse. We aimed to find markers of treatment response that might be suitable for routine use, using the gene expression profile of tumor tissue. Thirty one women with histologically-confirmed late-stage serous ovarian cancer were classified into 3 groups based on response to treatment (nonresponders, responders with relapse less than 12 months and responders with no relapse within 12 months). Gene expression profiles of these specimens were analyzed with respect to treatment response and survival (minimum 36 months follow-up). Patients' clinical features did not correlate with prognosis, or with specific gene expression patterns of their tumors. However women who did not respond to treatment could be distinguished from those who responded with no relapse within 12 months based on 34 gene transcripts (p < 0.02). Poor prognosis was associated with high expression of inhibitor of differentiation-2 (ID2) (p = 0.001). High expression of decorin (DCN) and ID2 together was strongly associated with reduced survival (p = 0.003), with an estimated 7-fold increased risk of dying (95% CI 1.9-29.6; 14 months survival) compared with low expression (44 months). Immunohistochemical analysis revealed both nuclear and cytoplasmic distribution of ID2 in ovarian tumors. High percentage of nuclear staining was associated with poor survival, although not statistically significantly. In conclusion, elevated expression of ID2 and DCN was significantly associated with poor prognosis in a homogeneous group of ovarian cancer patients for whom survival could not be predicted from clinical factors.
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Affiliation(s)
- Tanya R Newton
- Division of Population Health and Clinical Sciences, Queensland Institute of Medical Research, Brisbane, Australia
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10
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Le Pimpec-Barthes F, Danel C, Lacave R, Ricci S, Bry X, Lancelin F, Leber C, Milleron B, Fleury-Feith J, Riquet M, Bernaudin JF. Association of CK19 mRNA detection of occult cancer cells in mediastinal lymph nodes in non-small cell lung carcinoma and high risk of early recurrence. Eur J Cancer 2005; 41:306-12. [PMID: 15661557 DOI: 10.1016/j.ejca.2004.09.021] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2004] [Revised: 09/03/2004] [Accepted: 09/24/2004] [Indexed: 11/26/2022]
Abstract
This study was designed to screen occult cancer cells by CK19 mRNA detection using reverse transcriptase-polymerase chain reaction (RT-PCR) in mediastinal lymph nodes stations (MLNS) in non-small cell lung carcinoma (NSCLC). In 49 NSCLC patients free of mediastinal adenopathy on computed tomograph, 254 MLNS were evaluated by histopathology, immunohistochemistry (IHC) and RT-PCR. Of 225 non-tumoral MLNS on histopathology, 32 (14.2%) were positive by RT-PCR. IHC did not provide significant additional results. Seventeen patients were without mediastinal tumoral extension on histopathology and RT-PCR (Group 1), 16 were upgraded by RT-PCR (Group 2) and 16 pN2 on histopathology (Group 3). The two-year cancer-related death survival in Groups 1 (100%) and 2 (64.5%) was significantly different (P=0.04). The relative risk of recurrence in Group 2 compared with Group 1, evaluated by the Cox model multivariate analysis, was 5.61 (P=0.02). In conclusion, CK19 mRNA detected by RT-PCR in MLNS was significantly associated with an increased risk of rapid recurrence.
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11
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Saintigny P, Coulon S, Kambouchner M, Ricci S, Martinot E, Danel C, Breau JL, Bernaudin JF. Real-time RT-PCR detection of CK19, CK7 and MUC1 mRNA for diagnosis of lymph node micrometastases in non small cell lung carcinoma. Int J Cancer 2005; 115:777-82. [PMID: 15729695 DOI: 10.1002/ijc.20942] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Metastatic lymph nodes (LNs) are the major prognostic factor in resected non small cell lung carcinoma (NSCLC). However, almost 50% of pN0 patients relapse, suggesting metastatic cells undetected by current staging procedures. A combination of markers [cytokeratins 19 and 7 (CK19, CK7) and mucin type 1 (MUC1) mRNAs] was therefore evaluated by real-time RT-PCR in order to detect occult cancer cells. Forty-three NSCLC tumor samples, 4 micrometastatic, 6 metastatic and 84 histologically negative mediastinal LNs from 19 patients with NSCLC were evaluated as well as blood mononuclear cells from 29 healthy volunteers and 17 benign LNs. When tested on cell lines, RT-PCR was particularly efficient for evaluation of CK19, CK7 and MUC1 mRNA expression. All tumor samples were positive for at least 1 marker and 74% of samples were positive for all 3 markers. CK7 and CK19 mRNA were not detected in benign LN and blood cells from healthy donors in contrast with MUC1 mRNA. Only CK7 and CK19 mRNA were therefore used for evaluation of mediastinal LNs: the 6 histologically metastatic and the 4 micrometastatic LNs were positive for at least one marker. Among the 84 histologically negative LNs, 6 (7%) were positive for at least one marker, potentially changing the stage of 2 out of 19 patients. In conclusion, in our feasibility study, parallel molecular detection of CK19 and CK7 mRNA can be considered a specific diagnostic tool for the assessment of microscopic lymphatic spread. Its prognostic impact remains to be evaluated in a prospective study.
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Affiliation(s)
- Pierre Saintigny
- Histologie-Biologie Tumorale, UPRES EA 3499, Université Paris 6, Hôpital Tenon, Paris, France.
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12
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Schuster R, Max N, Mann B, Heufelder K, Thilo F, Gröne J, Rokos F, Buhr HJ, Thiel E, Keilholz U. Quantitative real-time RT-PCR for detection of disseminated tumor cells in peripheral blood of patients with colorectal cancer using different mRNA markers. Int J Cancer 2004; 108:219-27. [PMID: 14639606 DOI: 10.1002/ijc.11547] [Citation(s) in RCA: 97] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
The detection of disseminated tumor cells in peripheral blood from colorectal cancer patients by RT-PCR could be an attractive method for selecting patients for adjuvant therapy. We here report on real-time RT-PCR assays (LightCycler) to quantitate potential mRNA markers. We investigated specimens from colon carcinoma and normal colon mucosa tissues, cell lines, blood samples from 129 patients with colorectal cancer (all stages) and 58 reference blood samples (healthy donors, persons suffering from inflammatory bowel or infectious diseases). The expression profile in tissues showed high values for CEA and CK20, whereas in cell lines ProtM was predominant. All markers were detected in reference and patient blood samples (ProtM, 22, 17%; CEA, 84, 86%; CK20, 85, 88%). After quantitative analysis, the definition of cutoff values for each marker and the combination of markers, 13% of patients were judged to have elevated marker concentrations in their blood, from which only 6 had values significantly differing from cutoff value. There were no differences between stages of disease. In the case of 19 patients, investigated prior to and 1 week after surgery, 2 samples revealed a significant postoperative increase in CEA or CK20 mRNA concentration. In spite of high expression levels in tissues and cell lines, we were not able to differentiate satisfyingly mRNA markers originating from tumor cells and those from illegitimate transcription in hematopoetic cells in blood. We conclude that either copy numbers of analyzed markers in circulating tumor cells are not sufficient for detection or, more probably, peripheral blood is not a suitable compartment for detection of tumor cells in colorectal cancer.
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Affiliation(s)
- Ronny Schuster
- Department of Medicine III, University Hospital Benjamin Franklin, Free University Berlin, Berlin, Germany
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13
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Conzelmann M, Dieterle CP, Linnemann U, Berger MR. Cytokeratin 20 and guanylyl cyclase C mRNA is largely present in lymph node and liver specimens of colorectal cancer patients. Int J Cancer 2003; 107:617-28. [PMID: 14520701 DOI: 10.1002/ijc.11425] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The aim of our prospective study was to detect circulating epithelial cells (CEC) indicating the presence of disseminated tumor cells (DTC) in tissues affected by lymphatic and hematogenic colorectal cancer metastasis. DTC were tracked in lymph node, liver or bone marrow samples of 245 colorectal cancer patients using 2 independent RT-PCR assays for cytokeratin 20 (CK20) and guanylylcyclase C (GCC) that demonstrated a sensitivity of 1 colorectal cancer cell in 10(6) nucleated hematopoietic cells. CK20 mRNA was detected in 79% of lymph nodes, 35% of both liver lobes and 11% of bone marrow samples. GCC mRNA was found in 68% of lymph nodes, 60% of both liver lobes and 6% of bone marrow specimens. Both markers were recorded in 63% of lymph nodes, 45% of at least 1 liver lobe and 1% of bone marrow samples. There was no significant difference when comparing lymph node samples tested positive for both markers in patients with (N1/2; 65%) and without (N0; 56%) nodal involvement. The same was true when comparing the percentages of patients with and without clinically overt distant metastasis who were positive for both markers in at least 1 liver lobe (62% vs. 41%) or in bone marrow (4% vs. 0%). A score denoting the cumulative sum of tests indicating presence of CK20 and GCC mRNA in the liver was significantly related with UICC classification (p = 0.039). However, addition of lymph node results to this score decreased the correlation. The high incidence of clinically inconspicuous lymph node and liver samples tested positive for both markers emphasizes the function of these organs as primary filters for epithelial cells possibly shed from colorectal carcinomas. The potential prognostic significance of these findings warrants verification, especially regarding the importance of CEC or DTC resident in the liver of colorectal cancer patients.
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MESH Headings
- Aged
- Biomarkers, Tumor/genetics
- Biomarkers, Tumor/metabolism
- Bone Marrow/metabolism
- Bone Marrow/pathology
- Case-Control Studies
- Colorectal Neoplasms/metabolism
- DNA Primers/chemistry
- Female
- Gene Expression Regulation, Neoplastic
- Guanylate Cyclase/genetics
- Guanylate Cyclase/metabolism
- Humans
- Intermediate Filament Proteins/genetics
- Intermediate Filament Proteins/metabolism
- Keratin-20
- Liver/metabolism
- Lymph Nodes/metabolism
- Lymph Nodes/pathology
- Lymphatic Metastasis
- Male
- Neoplasm Staging
- Neoplastic Cells, Circulating/metabolism
- Prospective Studies
- RNA, Messenger/metabolism
- RNA, Neoplasm/genetics
- Receptors, Enterotoxin
- Receptors, Guanylate Cyclase-Coupled
- Receptors, Peptide/genetics
- Receptors, Peptide/metabolism
- Reverse Transcriptase Polymerase Chain Reaction
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Affiliation(s)
- Michael Conzelmann
- Unit of Toxicology and Chemotherapy, German Cancer Research Center, Heidelberg, Germany
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14
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Vlems FA, Ruers TJM, Punt CJA, Wobbes T, van Muijen GNP. Relevance of disseminated tumour cells in blood and bone marrow of patients with solid epithelial tumours in perspective. EUROPEAN JOURNAL OF SURGICAL ONCOLOGY 2003; 29:289-302. [PMID: 12711279 DOI: 10.1053/ejso.2002.1394] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Currently-used systems to predict prognosis in patients with solid epithelial tumours after surgical resection of the tumour do not give any guarantees for the individual patient. In this respect the clinical relevance of the presence of disseminated tumour cells in blood and bone marrow has been frequently studied. Because of growing awareness that information on merely the presence of disseminated tumour cells is not sufficient for prognostic and therapeutic purposes, attention for characterization of disseminated tumour cells has increased. Numerous reviews have already been published on the detection and clinical relevance of disseminated tumour cells. Therefore, this paper will mainly focus on the biological significance of these cells and discusses the (in)efficiency of the metastatic process, the genotypic and phenotypic characteristics of disseminated tumour cells, and their structure of appearance. Despite the fact that information gained on the several individual aspects is substantial, it did not render any solid solutions for individual patient management yet. Hence, a combined approach of several aspects of disseminated tumour cells together with characteristics and behaviour of the primary tumour is needed to substantially improve our knowledge on the role of disseminated tumour cells in the complex process of tumour metastasis.
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Affiliation(s)
- F A Vlems
- Department of Surgery, University Medical Centre Nijmegen, 6500 HB, Nijmegen, The Netherlands.
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15
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Vlems FA, Ladanyi A, Gertler R, Rosenberg R, Diepstra JHS, Röder C, Nekarda H, Molnar B, Tulassay Z, van Muijen GNP, Vogel I. Reliability of quantitative reverse-transcriptase-PCR-based detection of tumour cells in the blood between different laboratories using a standardised protocol. Eur J Cancer 2003; 39:388-396. [PMID: 12565993 DOI: 10.1016/s0959-8049(02)00631-7] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Differences in methods of reverse-transcriptase (RT)-polymerase chain reaction (PCR)-based detection of tumour cells in the blood gives rise to conflicting results, and standardisation is urgently needed. This pilot study aimed to assess the variation of RT-PCR-based detection of tumour cells in blood between four different laboratories using a commercially available kit with a standardised protocol. This kit allows comparison of results from different laboratories and facilitates the investigation of the influence of pre-analytical parameters. All laboratories analysed identical sets of blood samples spiked with tumour cells in a concentration range of 1-100 tumour cells/ml. To study at which level variation was introduced, three kinds of sample sets were generated in which (i) tumour cell RNA was spiked in the RNA of mononuclear cells (MNC), (ii) tumour cells were spiked in isolated MNC, and (iii) tumour cells were spiked in blood. Real-time quantitative RT-PCR was used to detect and quantify cytokeratin 20 (CK20) expression, which is indicative for the presence of epithelial tumour cells. All laboratories were able to detect CK20 expression in all spiked-RNA samples with limited variation in expression levels between laboratories. There was a positive correlation between the amount of spiked tumour cell RNA and CK20 expression level. RT-PCR analysis of spiked-MNC samples resulted in more variation in the CK20 expression levels between laboratories, however again all spiked samples were reported to be positive by all of the laboratories. The evaluation of spiked-blood samples gave rise to considerable quantitative and qualitative variation between the laboratories. Our results underline the importance and need for standardisation and extended quality control studies in the field of pre-analytics.
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Affiliation(s)
- F A Vlems
- Department of Surgery, University Medical Centre Nijmegen, PO-box 9101, 6500HB, Nijmegen, The Netherlands.
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16
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Tsavellas G, Huang A, McCullough T, Patel H, Araia R, Allen-Mersh TG. Flow cytometry correlates with RT-PCR for detection of spiked but not circulating colorectal cancer cells. Clin Exp Metastasis 2002; 19:495-502. [PMID: 12405286 DOI: 10.1023/a:1020350117292] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
The aim of this study was to determine whether flow cytometry (FACS) could detect spiked or circulating colorectal cancer cells. A flow cytometric assay was developed and its sensitivity compared with the reverse transcription polymerase-chain reaction (RT-PCR), using carcinoembryonic antigen (CEA) and cytokeratin (CK) 20 mRNA as target markers. Sensitivity limits for RT-PCR and flow cytometry (FACS) were established using spiked blood, and pre-operative blood samples from 20 colorectal cancer patients and 16 healthy no-cancer controls were analysed for circulating tumour cells (CTC) using both methods. Blood samples for FACS analysis were immuno-magnetically enriched using ferrofluid particles. CTC were defined as positive for pan-cytokeratin and negative for CD45 pan-leucocyte antigen (CK+/CD45- events). There was a significant (P < 0.0001) correlation between the number of spiked cancer cells and their recovery using FACS. The lowest detectable concentration was 20 spiked cancer cells in 14 ml blood for both RT-PCR and FACS. A positive FACS result significantly (P < 0.05) concurred with a positive RT-PCR result in spiked blood. The number of CK+/CD45- events detected in the blood of colorectal cancer patients was not significantly greater (P = 0.07) than in blood taken from 'no cancer' controls and furthermore there was no concordance (P = 1) between RT-PCR and FACS positivity in cancer patients' blood. FACS detection of tumour cells was feasible in vitro, and correlated with RT-PCR. However, its sensitivity in vivo was poor and did not correlate with RT-PCR detection of CTC. Uncertainties about antigen expression on normal circulating cells and about CTC phenotype need to be resolved, before FACS can be developed for detection of tumour cells within the circulation.
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Affiliation(s)
- G Tsavellas
- Division of Surgery, Faculty of Medicine, Imperial College School of Science, Technology and Medicine, Chelsea & Westminster Hospital, London, UK
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