HPV16-E6 mRNA is superior to Cytokeratin 19 mRNA as a molecular marker for the detection of disseminated tumour cells in sentinel lymph nodes of patients with cervical cancer by quantitative reverse-transcription PCR

Frauenklinik der Friedrich-Schiller-Universität Jena, Jena, Germany.
International Journal of Cancer (Impact Factor: 5.09). 05/2007; 120(9):1842-6. DOI: 10.1002/ijc.22521
Source: PubMed


About 10-15% of patients with cervical cancer suffer from recurrence despite histologically negative lymph nodes (pN0). Occult micrometastases or small tumour cell clusters may contribute to disease outcome. The aim of this study was to compare at the RNA level 2 known tumour-associated genes, HPV16-E6 and cytokeratin 19 (CK19), as molecular markers for the detection of disseminated tumour cells. Real-time reverse transcription PCR technology was used to quantify gene expression in histologically positive and negative sentinel lymph nodes (SLN) from 70 patients with cervical cancer. Lymph nodes from noncancer patients were used as controls. Calculated copy numbers were normalised to the geometric average of the most stable housekeeping genes. We observed a good correlation (R = 0.915) between the expression of both markers in SLN with histologically confirmed metastases. However, marker gene expression differed considerably in histologically negative nodes: CK19 transcripts were detected in 90 of 112 SLN (80.4%), whereas only 38 nodes (33.9%) were positive for HPV16 E6 mRNA. In particular, 62 of 74 SLN, which were negative by histology, and HPV16 E6 mRNA expressed CK19 mRNA. Moreover, 8 of 10 lymph nodes from noncancer patients expressed CK19 mRNA. Systematic errors due to RNA degradation or incomplete cDNA could be ruled out. It is concluded that HPV16 E6 mRNA is more specific and more sensitive for the detection of tumour cells in SLN than CK19 mRNA. The specificity of CK19 is limited because of low level expression in uninvolved pelvic lymph nodes.

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Available from: Mieczyslaw Gajda, May 01, 2015
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    • "Nevertheless, all methods rely on one or a combination of tumour cell specific markers. For virus-associated cancers i.e. cervical cancer viral transcripts are highly specific markers20. Furthermore, fusion transcripts present in different tumour entities or transcripts containing specific mutations could be established as tumour-specific markers. "
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    ABSTRACT: The detection of circulating tumour cells (CTC) in cancer patients may be useful for therapy monitoring and prediction of relapse. A sensitive assay based on HPV-oncogene transcripts which are highly specific for cervical cancer cells was established. The Digital-Direct-RT-PCR (DD-RT-PCR) combines Ficoll-separation, ThinPrep-fixation and one-step RT-PCR in a low-throughput digital-PCR format enabling the direct analysis and detection of individual CTC without RNA isolation. Experimental samples demonstrated a sensitivity of one HPV-positive cell in 500,000 HPV-negative cells. Spike-in experiments with down to 5 HPV-positive cells per millilitre EDTA-blood resulted in concordant positive results by PCR and immunocytochemistry. Blood samples from 3 of 10 CxCa patients each contained a single HPV-oncogene transcript expressing CTC among 5 to 15*10(5) MNBC. Only 1 of 7 patients with local but 2 of 3 women with systemic disease had CTC. This highly sensitive DD-RT-PCR for the detection of CTC may also be applied to other tumour entities which express tumour-specific transcripts. Abbreviations: CTC - circulating tumour cells, CxCa - cervical cancer, DD-RT-PCR - Digital-Direct Reverse Transcriptase PCR, HPV - Human Papilloma Virus, MNBC - mononuclear blood cells, ICC - immunocytochemistry.
    Scientific Reports 02/2014; 4:3970. DOI:10.1038/srep03970 · 5.58 Impact Factor
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    • "Only one showed expression of KRT19 transcripts , and its expression was at a very low level. On the other hand, Häfner et al. and Yuan et al. reported that HPV 16 E6 and SCCA mRNAs, respectively, were more suitable for the detection of tumor cells in SLNs from cervical cancer patients than KRT19 mRNA [22] [38]. They concluded that the KRT19 mRNA marker was expressed in normal LNs and was therefore not suitable for molecular detection of micrometastatic cervical cancer. "
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    ABSTRACT: The purpose of this study was to examine the utility of the one-step nucleic acid amplification (OSNA) assay using cytokeratin (CK) 19 (KRT19) messenger RNA (mRNA) for the detection of sentinel lymph node (SLN) metastases in cervical cancer patients. To determine a cutoff value, KRT19 mRNA was assessed by OSNA assay using 239 lymph nodes (LNs) (217 histopathologically negative LNs and 22 positive LNs). A cutoff value was determined by statistical analysis of the copy numbers obtained by OSNA assay. Subsequently, performance evaluation of the OSNA assay (applying the cutoff value above) on 130 SLNs (32 patients) was used to investigate (through concordance) whether the OSNA assay exhibited diagnostic performance equivalent to the two-mm interval histopathological examination. Two hundred fifty copies/μL of KRT19 mRNA in the OSNA assay appeared to be an optimal cutoff value. In performance evaluation of the OSNA assay, we identified five positive SLNs and 125 negative SLNs by OSNA assay using KRT19 mRNA, exhibiting 96.2% agreement with two-mm interval histopathological examination. Our results indicated that the KRT19 mRNA OSNA assay can detect LN metastases as accurately as two-mm interval histopathological examination and thus may be an effective additional or alternative method for rapid intra-operative examination of SLNs in cervical cancer.
    Gynecologic Oncology 06/2013; 130(3). DOI:10.1016/j.ygyno.2013.06.027 · 3.77 Impact Factor
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    • "Holmgren et al. suggested that some of these recurrences could be due to metastases not detected by routine H&E histology of lymph nodes, so-called "dormant" or "occult" metastases [8]. Hafner et al. reported that using routine H&E histology, the chances of identifying a tumour cell cluster of less than 3 cell diameters was only 1% [9]. "
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    ABSTRACT: Lymph node status is an important prognostic factor and a criterion for adjuvant therapy in uterine cancers. While detection of micrometastases by ultrastaging techniques is correlated to prognosis in several other cancers, this remains a matter of debate for uterine cancers. The objective of this review on sentinel nodes (SN) in uterine cancers was to determine the contribution of ultrastaging to detect micrometastases. Review of the English literature on SN procedure in cervical and endometrial cancers and histological techniques including hematoxylin and eosin (H&E) staining, serial sectioning, immunohistochemistry (IHC) and molecular techniques to detect micrometastases. In both cervical and endometrial cancers, H&E and IHC appeared insufficient to detect micrometastases. In cervical cancer, using H&E, serial sectioning and IHC, the rate of macrometastases varied between 7.1% and 36.3% with a mean value of 25.8%. The percentage of women with micrometastases ranged from 0% and 47.4% with a mean value of 28.3%. In endometrial cancer, the rate of macrometastases varied from 0% to 22%. Using H&E, serial sectioning and IHC, the rate of micrometastases varied from 0% to 15% with a mean value of 5.8%. In both cervical and endometrial cancers, data on the contribution of molecular techniques to detect micrometastases are insufficient to clarify their role in SN ultrastaging. In uterine cancers, H&E, serial sectioning and IHC appears the best histological combined technique to detect micrometastases. Although accumulating data have proved the relation between the risk of recurrence and the presence of micrometastases, their clinical implications on indications for adjuvant therapy has to be clarified.
    Journal of Experimental & Clinical Cancer Research 01/2010; 29(1):5. DOI:10.1186/1756-9966-29-5 · 4.43 Impact Factor
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