The Diagnostic Accuracy of Reverse Transcription-PCR Quantification of Cytokeratin mRNA in the Detection of Sentinel Lymph Node Invasion in Oral and Oropharyngeal Squamous Cell Carcinoma: A Comparison with Immunohistochemistry

University of Dundee, Dundee, Scotland, United Kingdom
Clinical Cancer Research (Impact Factor: 8.72). 05/2006; 12(8):2498-505. DOI: 10.1158/1078-0432.CCR-05-2136
Source: PubMed


The main goal of sentinel lymph node (SLN) detection in head and neck squamous cell carcinomas is to limit neck dissections to pN+ cases only. However, intraoperative + diagnosis cannot be routinely done using the current gold standard, serial step sectioning with immunohistochemistry. Real-time quantitative reverse transcription-PCR (RT-PCR) is potentially compatible with intraoperative use, proving highly sensitive in detecting molecular markers. This study postoperatively assessed the accuracy of quantitative RT-PCR in staging patients from their SLN.
A combined analysis on the same SLN by serial step sectioning with immunohistochemistry and quantitative RT-PCR targeting cytokeratins 5, 14, and 17 was done in 18 consecutive patients with oral or oropharyngeal squamous cell carcinoma and 10 control subjects.
From 71 lymph nodes examined, mRNA levels (KRT) were linked to metastasis size for the three cytokeratins studied (Pearson correlation coefficient, r = 0.89, 0.73, and 0.77 for KRT 5, 14, and 17 respectively; P < 0.05). Histopathology-positive SLNs (macro- and micrometastases) showed higher mRNA values than negative SLNs for KRT 17 (P < 10(-4)) and KRT 14 (P < 10(-2)). KRT 5 showed nonsignificant results. KRT 17 seemed to be the most accurate marker for the diagnosis of micrometastases of a size >450 mum. Smaller micrometastases and isolated tumor cells did not provide results above the background level. Receiver operating characteristic curve analysis for KRT 17 identified a cutoff value where patient staging reached 100% specificity and sensitivity for macro- and micrometastases.
Quantitative RT-PCR for SLN staging in cN(0) patients with oral and oropharyngeal squamous cell carcinoma seems to be a promising approach.

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    • "In this context, real-time reverse transcriptase-PCR (RT – PCR) may be helpful because it is operator independent and, with some adaptation, can be automated and rapidly performed (Raja et al, 2002; Hamakawa et al, 2004). In a previous study, we demonstrated that cytokeratin (CK)17 mRNA quantification could be evaluated in SLNs by semiquantitative RT – PCR and that neck staging could be performed with relevant sensitivity and specificity when compared with SS- IHC staging (Garrel et al, 2006). However, minute micrometastases (MIs) sized o450 mm produced signals similar to those of negative SLN controls and were thus undiagnosed by CK17 mRNA level quantification, showing the limitation of this mRNA marker for the detection of metastases in SLNs. "
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    ABSTRACT: Molecular diagnosis has been proposed to enhance the intra-operative diagnosis of sentinel lymph node (SLN) invasion in head and neck squamous cell carcinoma (HNSCC). Although cytokeratin (CK) mRNA quantification with real-time reverse transcriptase-PCR (QRT-PCR) has produced encouraging results, the more discriminating markers remain to be identified. Pemphigus vulgaris antigen (PVA), squamous cell carcinoma antigen (SCCA), and CK17 mRNA were quantified using QRT-PCR, and the results were compared with an extensive histopathological examination of the entire SLNs on 78 SLNs harvested from 22 patients with HNSCC. SCCA and CK17 quantification showed significantly higher mRNA values for macrometastases (MAs) than for either negative or isolated tumour cell (ITC) SLNs (P<0.01). Pemphigus vulgaris antigen allowed the discrimination of all MAs and micrometastases from both negative and ITC SLNs (P<0.001). For the neck staging of patients, considering metastatic vs non-metastatic status, receiver-operating characteristic curve analysis found areas under the curve of 93.8, 97.9, and 100% for CK17, SCCA, and PVA, respectively. With PVA, a cutoff value of 562 copies per 100 ng of cDNA permitted the correct distinction between patients with positive as opposed to negative neck nodes in all cases. PVA seems to be a highly promising marker for accurate intra-operative SLN staging in HNSCC by QRT-PCR.
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    ABSTRACT: Objective To assess the significance of sentinel lymph node biopsy (SLNB), serial section and cytokeratin immunohistochemical staining in the diagnosis and staging of Stage-cN0 oral squamous cell carcinoma (OSCC). Methods A blue stain, 99mTc-dextran SPECT lymphoscintigrapgy and intraoperative γ-ray probes were used to examine the sentinel nodes in 31 cases with Stage-cN0 oral cancer. The H&E staining and a cytokeratin AE1/AE3 immunohistochemistry (IHC) assessment, with serial sections, were conducted to provide results obtained from a routine pathological examination of lymph nodes. The value of the routine pathological examination of the sentinel lymph node (SLN), serial sections and IHC determination for cervical lymph node metastasis of Stage-cN0 OSCC was appraised. Results A total of 45, 55 and 51 SLNs were examined in 25 (80%), 31 (100%) and 30 (96.5%) of the cases, by using the blue stain, γ-ray probes, and SPECT lymphoscintigraphy, respectively. The average SLNs found in each case of the groups was 1.4 (1 to 3) and there were 1,302 non-NSLNs. Six positive SLN metastases were detected by routine pathological examination, among which 1 case was found to be an accompanied positive metastasis of non-SLN. One positive SLN metastasis was found after examination of serial sections plus routine H&E staining and 2 were detected using serial sections plus AE3 immunohistochemical staining methods. No positive NSLNs were found in the study. Conclusion In order to make more progress in accurate SLNB diagnosis, serial sections and IHC (AE1/AE3) methods can be used for examination of the micrometastases which are difficult to identify by routine pathological sections and H&E staining.
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