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

Abstract

Purpose: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. Experimental Design: 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. Results: From 71lymph 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 À4) and KRT 14 (P < 10 À2 ). KRT 5 showed nonsignificant results. KRT17 seemed to be the most accurate marker for the diagnosis of micrometastases of a size >450 Am. Smaller micrometastases and isolated tumor cells did not provide results above the background level. Receiver operating characteristic curve analysis for KRT17 identified a cutoff value where patient staging reached 100% specificity and sensitivity for macro-and micrometastases. Conclusion: Quantitative RT-PCR for SLN staging in cN 0 patients with oral and oropharyngeal squamous cell carcinoma seems to be a promising approach.The extent of lymph node involvement in clinically and computed tomography scan N 0 (cN 0 ) oral and oropharyngeal squamous cell carcinomas is f30% to 40% and is one of the main prognostic factors (1). Until recently, a neck dissection was advocated routinely both to assess nodal involvement and to remove occult minimal residual cancer (2). A more recent approach consists of limiting lymph node surgery to a staging procedure by taking only the sentinel lymph nodes (SLN) which are representative of the whole neck node system (3). Such a strategy aims to permit more thorough analysis of only a few lymph nodes to enhance the sensitivity and the specificity of the diagnosis of lymph node invasion and thus select only pN+ patients on whom to perform a neck dissection. Intraoperative diagnosis must therefore have a predictive negative value close to 100%. At present, many methods of SLN analysis are used. The reference method for pathologic analysis is histopathologic examination by serial step sectioning with immunohistochemistry, staining with anticytokeratin antibodies in cases of squamous cell carcinoma (4, 5). Such analysis is able to diagnose three levels of nodal invasion (6): isolated tumor cells <0.2 mm, micrometastases V2 mm, and macromet...

Cytokeratin 17 mRNA expression has potential for diagnostic marker of oral squamous cell carcinoma

“…Detection of metastases using molecular markers has been reported previously (Roepman et al 2005). However, there is still a need for additional sensitive markers to detect individual DTCs themselves in diVerent specimens (Garrel et al 2006). The Wrst step in identifying those candidates is to determine which genes are frequently and strongly expressed in primary site of OSCC by high-density cDNA microarrays allowing the investigation of a large number at the same time.…”

Quantitative expression study of four cytokeratins and p63 in squamous cell carcinoma of the tongue: suitability for sentinel node navigation surgery using one-step nucleic acid amplification

“…Single frozen sections can be obtained intra-operatively, but also show a suboptimal sensitivity of between 50% and 85% 6 7. Therefore, one-step nucleic acid amplification (OSNA) has recently been introduced for breast cancer using cytokeratin 19 (CK19), and various authors have reported that OSNA assay8–10 can detect lymph node metastasis as accurately as conventional pathology, allowing immediate diagnosis of lymph node metastasis and eliminating the need for pathological evaluation during surgery.…”

Pemphigus vulgaris antigen mRNA quantification for the staging of sentinel lymph nodes in head and neck cancer

“…Indeed, this rapid technique seems to be a valuable tool for the detection of lymph node invasion of breast adenocarcinomas (Kataoka et al, 2000), skin melanomas (Blaheta et al, 2001), and HNSCC (Hamakawa et al, 2000(Hamakawa et al, , 2004Becker et al, 2004;Shores et al, 2004). We have previously shown that CK17 mRNA quantification could accurately determine sentinel node staging (Garrel et al, 2006). However, this marker did not permit the detection of MIs sized o450 mm.…”

“…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.…”