The assessment of the robustness of microRNAs from oral cytological scrapings

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MicroRNAs have been associated with almost all types of human malignancies including hematological 1 and solid tumors 2. Differential microRNA expression has been found in both oral premalignant and malignant cells 3. Hence, there is a need for the investigation of microRNA expression in potentially malignant oral mucosal lesions as well as oral squamous cell carcinoma (OSCC) as deregulated microRNAs may be an early reliable marker for malignancy as well as a potential target for cancer prevention.
One of the factors limiting the OSCC detection rate is the reluctance of practitioners and patients to have repeat scalpel biopsies performed on suspect lesions 4. Thus, although the developed method to assess oral cancer risk using microRNA is objective, it may not greatly improve OSCC detection rates as it provides no impetus for increased lesion sampling in the clinic.
Over the past years, there has been renewed interest in oral cytology as an adjuvant clinical tool in the investigation of oral mucosal lesions. Several studies have shown encouraging results with oral brush cytology 5. However, there has been varying evidence cited with regard to the accuracy, sensitivity and specificity of OralCDx compared to routine scalpel biopsy that has not supported the use of OralCDx in a routine manner 7. Navone et al. 10 compared the histopathology of cells obtained using brush biopsy, exfoliation using a dermatologic curette to conventional scalpel biopsy. The smears from the conventional cytology group were deemed inadequate in 12.4% of the samples (fewer than 30 well‐preserved cells from the intermediate or parabasal–basal epithelial layer), whereas only 8.8% were deemed inadequate in the dermatologically curetted cytology group. A prospective study carried out on 164 patients with premalignant lesions (PML) comparing scalpel biopsy and microbiopsy with a dermatological curette further supported the use of microbiopsy 6. Microbiopsy diagnosis was in agreement with scalpel biopsy in 91.14% of the cases 6. Its higher sensitivity levels than scalpel biopsy (97.65% vs. 85.88%) and its high negative predictive value (97.33%) suggest it to be an effective tool for diagnosis of PML 6.
It has been demonstrated using comparison between FFPE and frozen tissues that microRNA is robust and preserved in scalpel biopsies 2. Tissue sampling via scalpel biopsy damages the layer of cells at the incision points but also provides a pool of cells within the tissue that would be undamaged by the action of the scalpel. Thus, the RNA obtained from a scalpel biopsy would be a mixture of degraded and intact material. Cytological sampling using a curette may produce a greater proportion of damaged cells than biopsy which could impact comparative microRNA quality. To make a cytological scraping method viable for use in the general clinical setting, the mode of sample storage and transport to a diagnostic laboratory also needs to be considered as the parameters of storage (transport) medium; temperature and time could also have an impact on the data obtained.
Ibberson et al. 12 conducted an extensive study where microRNA abundance was assessed using microarray analysis or microRNA‐specific real‐time quantitative PCR (miQPCR) and tissue samples that were maintained on ice for defined periods of time (and thus slowly degraded) prior to RNA extraction. The conclusion of this study was that the loss of RNA integrity leads to unpredictable microRNA profiles for both array‐based and miQPCR assays 12. However, scrutiny of these results show that even after degradation of RNA over a span of 2 h, there is only a 2–4 unit CT change in microRNA levels 12.
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