**5. In Situ Hybridization (ISH)**

positively in tubal metaplasia. In practice, it is not usually necessary to undertake a count of positive cells, as there are typically only scattered positive nuclei in tubal metaplasia, while

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

All cases of adenocarcinoma in situ showed markedly increased Ki-67 labeling together with diffuse nuclear and cytoplasmic reactivity for p16 in the study by Little and Stewart [111], and typically the positive cells were sharply demarcated from the adjacent normal, unstained

However, AIS may occasionally exhibit a low proliferation index [110]. Additionally, some benign lesions, like endometriosis, may show a high proliferation index. Thus, a combination of different markers is more useful than isolated stains in the evaluation of glandular intrae‐

Bcl-2 is a member of a large family of proteins, some inhibiting and others favoring apoptosis. Lesions of adenocarcinoma in situ with significant apoptosis show negative or focally positive bcl-2 immunostains [109]. In the contrary, tubal metaplasia and endometriosis typically exhibit diffuse cytoplasmic positivity, reminiscent of normal fallopian tube epithelium and prolifer‐ ative endometrium [20]. However, normal endocervical glands are also negative. Consequent‐ ly, immunostaining for bcl-2 can comprise part of a panel of antibodies, including p16 and Ki-67 [20,112]. It can also be used in the evaluation of cauterized margins [20], as already

Except for the above discussed markers, several other immunostains have been reported to be of help in the diagnosis of glandular lesions. Vimentin can be useful in distinguishing AIS from endometriosis and tuboendometrial metaplasia, since the latter two entities exhibit cytoplas‐ mic positivity. They also show positivity for estrogen receptor, while AIS is negative or focally positive [20]. One additional marker is carcinoembryonic antigen (CEA), which is observed cytoplasmically in a significant percentage of AIS cases, whereas normal endocervical

Interestingly, complete negativity for cyclin D1 was commonly observed in AIS in a study by Little and Stewart [111], in contrast to tuboendometrial metaplasia and normal endocervical epithelia, although staining was typically focal in the latter. Microglandular hyperplasia and mesonephric duct elements were also cyclin D1 positive although relatively few cases were

It should be also noted that the intestinal type of AIS has been reported to show CK7 positivity and CK20 negativity or extremely focal positivity, in spite of the presence of morphological

Finally, immunohistochemical stains for cytomegalovirus and herpesvirus can be of use for confirmationoftheseinfections,althoughtheyarenotusuallylikelytobeconfusedwithAIS[34].

intestinal differentiation in a few cases examined, as reported by McCluggage [20].

the majority of nuclei are positive in AIS [20].

discussed for p16 and Ki-67 in squamous lesions.

epithelium shows only luminal or no staining.

endocervical epithelium.

pithelial alterations.

*4.2.4. Other markers*

examined in that study.

*4.2.3. bcl-2*

Detection of HPV nucleic acids is performed by methods that can be broadly subdivided into: a) methods based on target amplification, and b) those based on signal amplification [113]. In addition to several existing liquid phase techniques, in situ hybridization (ISH) methods have been developed for the detection of nucleic acids in cytological and histolog‐ ical specimens. Efforts at improving ISH performance have focused both on amplifying nucleic acid targets before hybridization or on amplifying signals afterwards (e.g. by using in situ PCR, or tyramide signal amplification). Both fluorescent detection and coloured substrate deposition followed by bright-field microscopy can be used, and can be com‐ bined with tyramide signal amplification. In addition, ISH assays can be automated along the same lines as immunohistochemistry.

Issues concerning sensitivity of ISH techniques in comparison to PCR have been repeatedly raised. However, these techniques are becoming increasingly sensitive [114,115]. One main contribution of ISH to HPV research is the fact that it permits concurrent morphological evaluation of the cells examined, especially in the case of histological specimens, which is a significant advantage in comparison to liquid phase techniques. In addition, the signal patterns observed in HPV in situ hybridization have been reported to be associated with the physical status of viral DNA in the cell,that is episomal or integrated. Specifically, the punctate pattern of positivity has been linked to viral forms integrated in the host genome [86,116-118].

In a study by Kalof et al. punctate signals were detected in 17/17 (100%) CIN 2/3 lesions, but in only 13.6% of high-risk HPV-positive CIN 1 lesions [86]. In cytology material Ho et al. reported a punctate pattern in 8.7% of CIN1 lesions vs 34.0% of CIN3 lesions [119].

ISH and PCR had fair to good agreement in detecting HPV DNA across CIN categories in a study by Guo et al.; however, ISH detected significantly fewer HPV-positive cases in carcino‐ mas than PCR did, probably as a result of lower copy numbers of episomal HPV DNA in the latter [120]. In addition, although the pure punctate pattern of HPV indicated a high level of viral integration, in cases with mixed signal patterns the level of HPV integration could not be accurately determined, probably due to a variation in the percentage of the two patterns in these cases.

According to Kong et al., in cases of atypical squamous metaplasia, p16 reactivity (focal strong and diffuse strong) was significantly more sensitive than ISH in correlating with the presence of human papillomavirus as detected by PCR [121]. Voss et al. compared a fluorescence in situ hybridization (FISH) HR-HPV assay to Hybrid Capture 2 (HC2) and PCR for the detection of HR-HPV subtypes in cervical cytology specimens [122]. FISH was concordant with HC2 and PCR in 85% and 82% of the specimens, respectively, while HC2 and PCR were concordant in 84% of the specimens. In a more recent study by Kelesidis et al., ISH exhibited a sensitivity of 89.5% for the detection of CIN2+ lesions, while PCR showed sensitivity of 94.7% for these lesions. Importantly, a percentage of ISH-positive cases was not detected by polymerase chain reaction (performed on liquid-based sample media), emphasizing the technical problems and limitations of the techniques [114].

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 evaluation of the area examined is necessary.

**7. Conclusions**

dressed by the current vaccines.

Evanthia Kostopoulou and George Koukoulis

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Pathology Department, Faculty of Medicine, University of Thessaly, Greece

of the breast and female genital organs. Lyon, France: IARC; 2003

smear to the vaccine era. J. Clin. Oncol. 2003;21(10 Suppl): 224–30.

carcinomas. Curr. Top. Microbiol. Immunol. 1977;78: 1–30.

[1] Wells M, Ostor A, Crum C, Franceschi S et al. Tumours of the uterine cervix. Epithe‐ lial tumours. In: Tavassoli FA, Devilee P, eds. WHO Classification of tumors: Tumors

[2] Crum CP, Abbott DW, Quade BJ. Cervical cancer screening: from the Papanicolaou

[3] zur Hausen H. Papillomaviruses—to Vaccination and Beyond. Biochemistry (Mos‐

[4] zur Hausen H. Papillomaviruses in the causation of human cancers — a brief histori‐

[5] zur Hausen, H.. Human papilloma viruses and their possible role in squamous cell

**Author details**

**References**

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.

Ancillary Techniques in the Histopathologic Diagnosis of Squamous and Glandular Intraepithelial Lesions…

http://dx.doi.org/10.5772/55897

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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‐

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

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 and should be considered in the application of these techniques.

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 multiple infections [127].

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 alone.
