*4.1.4. ProEx C*

In conclusion, cyclin E immunostaining could be used to discriminate reactive from neoplastic epithelium [14] especially in conjunction with other markers, discussed in other parts of the present text. However, it is not useful in the distinction of low-grade from high-grade lesions.

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

Ki-67 is an antigen expressed in the nuclei of proliferating cells during the whole cell cycle, except for the G0 and G1 early phases. MIB-1 is a monoclonal antibody that detects this antigen in paraffin tissue sections. Although positivity is observed under normal conditions in the lower compartments of the multilayered squamous epithelium, staining of the middle and

Immunopositivity for Ki-67 increases as a function of increasing lesion grade [16,19,95-98]. HSIL/CIN3 lesions usually show nuclear positivity scattered throughout all epithelial layers, while lower grade lesions show less diffuse immunoreaction. However, immunostains should be interpreted with caution. It is well-known to pathologists that reactive and reparative changes may pose a problem in the examination of cell proliferation, and, in the case of Ki-67, immunostaining may extend through the upper layers of the epithelium. In addition, tangen‐ tial sectioning may sometimes result in the false impression that positive parabasal cells are

It should be noted that Ki-67 immunohistochemical stain may be especially helpful in differ‐ entiating atrophic epithelial changes from high-grade lesions [14]. It can also be used in the evaluation of cauterized margins, which often pose diagnostic problems [20]. In addition, Ki-67 immunostaining can be used as an adjunct to other markers. In one study of metaplastic cervical epithelia, addition of Ki67 positivity in >50% of lesional cells to p16 "band" positivity increased specificity (from 92.5 to 96%) and positive predictive value (from 85.7 to 91.7%) for the diagnosis of high-grade CIN [100]. Dual IHC stain with evaluation of colocalization of

upper layers is indicative of an intraepithelial lesion (Figure -5).

more superficially placed, thus leading to a SIL diagnosis [99].

antibodies represents an interesting approach to this theme [101].

(a) (b)

**Figure 5.** Ki-67 positivity in a low-grade SIL.

*4.1.3. Ki67*

ProEx C is a recently developed biomarker reagent that targets two proteins having a signifi‐ cant role in the regulation of DNA replication during S-phase: the minichromosome mainte‐ nance protein 2 (MCM2) and DNA topoisomerase IIa (TOP2A). TOP2A is a nuclear enzyme that regulates the enzymatic unlinking of DNA strands during chromosome replication. MCM2 functions also during DNA replication by loading the pre-replication complex onto DNA and unwinding the latter through helicase activity to permit synthesis. These proteins are overexpressed in aberrant S-phase induction and have been shown to be overexpressed in CINs and cervical carcinomas [98,99,102,103]. ProEx C appears to be efficient in distinguishing reactive epithelial changes from squamous lesions. The stain can be used alone or in conjunc‐ tion with p16.

The staining pattern of ProEx C in histologic sections is evaluated in a way reminiscent of MIB-1, since only nuclear positivity above the basal one-third layer is considered positive [99]. ProEx C has been reported to stain reactive epithelial cell nuclei less and LSIL nuclei more than MIB-1 [99]. Strong positive staining for ProEx C involving the lower and upper halves of the epithelium was observed in 92% of high-grade squamous intraepithelial lesions in a study by Badr et al. [102]. Condylomas and CIN I showed greater variability in patterns of staining, with immunopositivity extending into the upper half of the epithelium in 48% of cases. According to the authors, the stain can be applied to confirm the diagnosis of HSIL and to triage cases of atypical squamous metaplasia. According to Shi et al. [103], ProEx C is a better marker than p16 for the detection of LSILs, showing positivity in 94% of the cases in a series of 34 LSILs.

One study by Pinto et al. [98] examined cases with the differential diagnosis of HSIL vs reactive epithelial changes. ProEx C showed 87% sensitivity and 71% specificity for SIL in biopsy material. The authors reported a larger number of cells stained by ProEx C in comparison to MIB-1 in both HSIL and LSIL cases. In addition, the combination of p16 and ProEx C predicted more NoSIL (including normal, reactive, and/or atrophic epithelia) than p16 and MiB-1 (61% vs 43%). These observations suggested that ProEx C could be more useful in the distinction of reactive epithelial changes from SILs than MiB-1.

Sanati et al. reported a sensitivity and specificity of 89% and 100%, respectively, for ProExC immunostain in distinguishing high-grade squamous intraepithelial lesions from squamous metaplasia, while positive and negative predictive values were 100% and 82%, respectively [104]. In a recent study by Guo et al., diffuse positivity for ProExC significantly increased from benign cervix/CIN 1 to CIN 2 or 3/carcinoma, while the highest specificity for CIN 2+ and CIN3+ (100% and 93%, respectively) was achieved when immunostaining was positive for both ProExC and p16, suggesting that it is advantageous to use these two markers together in order to distinguish high-grade lesions from their mimics [105]. The use of the same two markers, p16 and ProExC, as a first step, followed by Ki-67 immunostaining in discordant cases, has been suggested as cost saving strategy by Walts and Bose [106]. According to these authors, performing the two above stains initially and adding Ki-67 only when p16 and ProExC yield discordant results provided the same diagnostic accuracy while reducing the cost, since only one third of the cases required performance of the third stain.

### *4.1.5. L1 capsid protein*

HPV L1 capsid protein is the major structural protein of human papillomavirus. In the last few years L1 immunoreactivity has been examined repeatedly in cytologic material. Nuclear positivity is mainly observed in productive lesions and is gradually lost in high grade lesions and carcinomas.

**4.2. Immunohistochemical stains in the diagnosis of glandular intraepithelial lesions**

and Herpesvirus. Biomarkers can be of help in these cases.

be observed in tubal metaplasia; however, it is commonly focal/weak.

problematic cases in conjunction with careful morphologic examination.

*4.2.1. p16*

labeling in >25% of cells.

between normal and neoplastic epithelium.

*4.2.2. Ki-67*

Several entities have to be distinguished from AIS, including cervical endometriosis, tubal metaplasia, reparative changes, Arias-Stella reaction, atypia due to irradiation, atypical forms of microglandular hyperplasia, as well as other viral infections, specifically Cytomegalovirus

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Already discussed in the paragraphs concerning squamous intraepithelial lesions, p16 has also emerged as a potentially useful marker in the evaluation of glandular cervical alterations [20, 109-111]. Most AIS lesions show diffuse nuclear and/or cytoplasmic immunopositivity (Figure 6). Microglandular hyperplasia and reactive lesions are usually negative for p16. Positivity can

p16 was diffusely and strongly expressed (3+) in 29/29 AIS in a study by Negri et al [110], while patchy positivity was observed in tubal metaplasias, endometriosis and endometrial samples. Likewise, all 19 AIS cases showed diffuse nuclear and cytoplasmic reactivity in a study by Little and Stewart [111], while rare single cells were positive in normal endocervical epitheli‐ um. Staining was focal in most cases of tubo-endometrioid metaplasia in this study; however, eight of these latter cases included glands that were diffusely p16 positive and/or showed Ki-67

These results emphasize the importance of using panels of antibodies in some of these

**Figure 6.** p16 immunopositivity in AIS. Compare with adjacent normal glands. Abrupt transition is focally observed

Nuclear positivity for Ki-67 is usually observed in >30% of cells in adenocarcinoma in situ, and often in the majority of the cells. In the contrary, only a small percentage of cells (<10%) stain

It has been suggested that combined L1/p16 IHC may be helpful for clinical management, especially in cases in which the grade of the lesion is difficult to assess [91]. In a study by Negri et al. L1 was expressed in 34.85% of CIN1 cases, including 12/38 (31.56%) cases with coexisting CIN3 in conization specimens and 11 of 28 (39.29%) biopsy specimens from women with cytologically proven regression [91]. In the latter group a high staining score was often observed. The authors suggested that combining p16 and L1 immunostaining might allow for a distinction between different risk patterns of LSIL.

In a study by Galgano et al. immunohistochemical staining of HPV L1 was negatively associated with the increasing severity of their consensus diagnosis (ptrend<0.001) and decreased with increasing intensity of p16INK4a (ptrend<0.001) and Ki-67 (ptrend<0.001) [107]. Positivity for L1 was observed in 32%, 32.2%, and 16.5% of CIN1, CIN2, and CIN3/AIS, respectively, and was also observed in 3.3% of negative specimens. It was negatively associated with having a CIN2+ or CIN3+ diagnosis (OR=0.62, and 0.18, respectively). The authors reported that L1 IHC detection was neither sensitive nor specific for any group of cervical neoplasia in biopsy material and this was attributed to the complexity of the temporal evolution of the HPV virion production which may be quite transient. It is interesting that L1 positive cases with a negative consensus diagnosis in this study commonly had at least 1 reviewer diagnosis of CIN1, revealing once again the difficulties in the distinction of SIL vs negative for SIL.

In a recent study by Gatta et al. L1 was expressed in the nuclei of superficial cells of dysplastic epithelia, often with characteristics of koilocytes [108]. L1 positivity was observed in 8/32 CIN1 biopsies (25%) and in 1/10 CIN2 cases (10%), but it was not observed in CIN3 and carcinoma cases examined. Their only case which showed a punctate signal with catalyzed signalamplified colorimetric DNA in situ hybridization, suggestive of viral integration, belonged to the L1-negative/p16-positive group.

#### *4.1.6. Other markers*

In the above paragraphs some of the most important markers used in the histopathologic diagnosis of SIL have been presented. Ki67 and p16 have been used for several years in many laboratories worldwide, while ProEx C and L1 have only been in use for the last few years. However, several other markers have been tested in cervical biopsies for their potential utility in diagnosis. It should be noted that in recent years development of high-throughput technol‐ ogies, as gene expression profiling, has increased the potential for biomarker discovery. However, although some of these markers showed promising results, in most cases they did not present any specific advantages in comparison to the already existing biomarkers from a diagnostic point of view.

## **4.2. Immunohistochemical stains in the diagnosis of glandular intraepithelial lesions**

Several entities have to be distinguished from AIS, including cervical endometriosis, tubal metaplasia, reparative changes, Arias-Stella reaction, atypia due to irradiation, atypical forms of microglandular hyperplasia, as well as other viral infections, specifically Cytomegalovirus and Herpesvirus. Biomarkers can be of help in these cases.

### *4.2.1. p16*

*4.1.5. L1 capsid protein*

and carcinomas.

a distinction between different risk patterns of LSIL.

the L1-negative/p16-positive group.

*4.1.6. Other markers*

diagnostic point of view.

HPV L1 capsid protein is the major structural protein of human papillomavirus. In the last few years L1 immunoreactivity has been examined repeatedly in cytologic material. Nuclear positivity is mainly observed in productive lesions and is gradually lost in high grade lesions

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

It has been suggested that combined L1/p16 IHC may be helpful for clinical management, especially in cases in which the grade of the lesion is difficult to assess [91]. In a study by Negri et al. L1 was expressed in 34.85% of CIN1 cases, including 12/38 (31.56%) cases with coexisting CIN3 in conization specimens and 11 of 28 (39.29%) biopsy specimens from women with cytologically proven regression [91]. In the latter group a high staining score was often observed. The authors suggested that combining p16 and L1 immunostaining might allow for

In a study by Galgano et al. immunohistochemical staining of HPV L1 was negatively associated with the increasing severity of their consensus diagnosis (ptrend<0.001) and decreased with increasing intensity of p16INK4a (ptrend<0.001) and Ki-67 (ptrend<0.001) [107]. Positivity for L1 was observed in 32%, 32.2%, and 16.5% of CIN1, CIN2, and CIN3/AIS, respectively, and was also observed in 3.3% of negative specimens. It was negatively associated with having a CIN2+ or CIN3+ diagnosis (OR=0.62, and 0.18, respectively). The authors reported that L1 IHC detection was neither sensitive nor specific for any group of cervical neoplasia in biopsy material and this was attributed to the complexity of the temporal evolution of the HPV virion production which may be quite transient. It is interesting that L1 positive cases with a negative consensus diagnosis in this study commonly had at least 1 reviewer diagnosis of CIN1,

In a recent study by Gatta et al. L1 was expressed in the nuclei of superficial cells of dysplastic epithelia, often with characteristics of koilocytes [108]. L1 positivity was observed in 8/32 CIN1 biopsies (25%) and in 1/10 CIN2 cases (10%), but it was not observed in CIN3 and carcinoma cases examined. Their only case which showed a punctate signal with catalyzed signalamplified colorimetric DNA in situ hybridization, suggestive of viral integration, belonged to

In the above paragraphs some of the most important markers used in the histopathologic diagnosis of SIL have been presented. Ki67 and p16 have been used for several years in many laboratories worldwide, while ProEx C and L1 have only been in use for the last few years. However, several other markers have been tested in cervical biopsies for their potential utility in diagnosis. It should be noted that in recent years development of high-throughput technol‐ ogies, as gene expression profiling, has increased the potential for biomarker discovery. However, although some of these markers showed promising results, in most cases they did not present any specific advantages in comparison to the already existing biomarkers from a

revealing once again the difficulties in the distinction of SIL vs negative for SIL.

Already discussed in the paragraphs concerning squamous intraepithelial lesions, p16 has also emerged as a potentially useful marker in the evaluation of glandular cervical alterations [20, 109-111]. Most AIS lesions show diffuse nuclear and/or cytoplasmic immunopositivity (Figure 6). Microglandular hyperplasia and reactive lesions are usually negative for p16. Positivity can be observed in tubal metaplasia; however, it is commonly focal/weak.

p16 was diffusely and strongly expressed (3+) in 29/29 AIS in a study by Negri et al [110], while patchy positivity was observed in tubal metaplasias, endometriosis and endometrial samples. Likewise, all 19 AIS cases showed diffuse nuclear and cytoplasmic reactivity in a study by Little and Stewart [111], while rare single cells were positive in normal endocervical epitheli‐ um. Staining was focal in most cases of tubo-endometrioid metaplasia in this study; however, eight of these latter cases included glands that were diffusely p16 positive and/or showed Ki-67 labeling in >25% of cells.

These results emphasize the importance of using panels of antibodies in some of these problematic cases in conjunction with careful morphologic examination.

**Figure 6.** p16 immunopositivity in AIS. Compare with adjacent normal glands. Abrupt transition is focally observed between normal and neoplastic epithelium.

#### *4.2.2. Ki-67*

Nuclear positivity for Ki-67 is usually observed in >30% of cells in adenocarcinoma in situ, and often in the majority of the cells. In the contrary, only a small percentage of cells (<10%) stain 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 the majority of nuclei are positive in AIS [20].

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

the same lines as immunohistochemistry.

these cases.

limitations of the techniques [114].

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

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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.

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

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

reported a punctate pattern in 8.7% of CIN1 lesions vs 34.0% of CIN3 lesions [119].

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 endocervical epithelium.

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‐ pithelial alterations.

#### *4.2.3. bcl-2*

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 discussed for p16 and Ki-67 in squamous lesions.

#### *4.2.4. Other markers*

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 epithelium shows only luminal or no staining.

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 examined in that study.

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

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