**7. Conclusions**

As is apparent from the above results, the applications of HPV DNA ISH are partly dependent on the sensitivity of the assay and its sufficiency to carry a high negative predictive value [14]. This is especially important if clinical decisions are based on a negative result. However, ISH represents a useful tool for ancillary molecular HPV testing when concurrent morphological

136 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

Except for HPV nucleic acids, there are also other applications of in situ hybridization techniques in cervical specimens, including the detection of amplification of the gene coding for the telomerase RNA component (TERC) at 3q26 [56,123,124]. TERC amplification has been reported to increase with severity of dysplasia and it has been suggested that this might serve

Except for DNA ISH, in situ RNA detection shows promising results in certain applications. RNA markers have emerged as an important group of biomarkers after the widespread use of genome-wide gene expression profiling techniques. New sensitive RNA in situ hybridization methods can detect more than one target simultaneously, can be applied on formalin-fixed paraffin-embedded tissue, and they allow for simultaneous evaluation of morphology [125]. As expected, preanalytical variables related to fixation can affect biomarker measurements

Both cellular and viral proteins related to lesion prognosis, previously examined at the mRNA level with PCR techniques, might prove to be important markers for RNA ISH applications. Recently, transcriptional analysis of HPV16 genes by in situ hybridization in histological sections of cervical dysplasia revealed transcription patterns that bring into question some of the current beliefs on the mechanism of HPV-16 infection in the progression to cervical cancer [126]. The detection of HPV E6/E7 mRNA expression appears also promising in the case of

In addition, the polymerase chain reaction has been used for target amplification before hybridization, with promising results. *In situ* PCR combines increased sensitivity with the anatomical localization provided by in-situ hybridization, and allows the examination of the specific genetic material at a specific cellular level. Although the method is subject to both false positive and negative outcomes, it allows correlation of viral DNA localization with relevant target proteins, thus providing important information concerning the development of cervical neoplasia [128,129], which cannot be obtained by polymerase chain reaction-based methods

Finally, it should be noted that new techniques continue to emerge in the field of cervical lesion detection. Of particular interest in the context of biopsy processing is the development of a water-soluble protein-saving biopsy processing method, which is followed by analysis of proteins of the supernatant samples. This method resulted in the identification of proteins that discriminate between grades of cervical neoplasia, while preserving the tissue for conventional

evaluation of the area examined is necessary.

multiple infections [127].

**6. Other techniques**

microscopic analysis [130,131].

alone.

as a marker in the distinction between low- and high-grade lesions.

and should be considered in the application of these techniques.

In the above text important ancillary techniques currently in use in several laboratories worldwide for the evaluation of cervical biopsies have been presented, along with their main applications in diagnosis. Suggested panels of antibodies for specific diagnostic dilemmas have been discussed. Furthermore, the significance of certain negative stains has been presented. It should be noted that histopathologic examination remains the ''gold standard'' for the diagnosis of low- and high-grade SIL and AIS; however, certain biomarkers have emerged as helpful adjuncts, assisting in a more precise diagnosis of cervical precursor lesions.

It is obvious from the above presented data that the diagnosis of a lesion in a diagnostically challenging case cannot at present be based solely on any particular marker, but rather on a combination of markers with careful morphologic evaluation. Important requirements for the proper use of the presented markers include standardization of protocols and familiarity with the patterns of immunostaining. Finally, an important issue, not specifically analyzed, which may emerge in the next few years and merits further study, is the exact performance of these markers in the detection of lesions related to uncommon HPV types, other than those ad‐ dressed by the current vaccines.
