**4. Immunohistochemical stains in the diagnosis of cervical intraepithelial lesions**

The role of the pathologist examining a cervical biopsy suspicious for an intraepithelial lesion consists of: a) confirming or excluding the presence of a lesion, b) excluding other entities entering the differential diagnosis, and, c) if the diagnosis of an intraepithelial lesion is confirmed, distinguishing a low-grade from a high-grade alteration. Histopathological diagnosis of intraepithelial lesions is based on well-defined criteria, as discussed above. However, in certain cases distinguishing both low- and high-grade lesions from their mimics may pose problems [14,61], even to experienced gynecologic pathologists. Florid reactive changes and metaplastic patterns may present histopathologic features, which may cause difficulties in the distinction from HPV-induced alterations. Attempts have been made to redefine the traditional criteria for lesion diagnosis, while other efforts aimed at the adoption of new, more objective methods, which might support the former [22,23,62-64]. However, studies attempting to correlate HPV presence and replication to certain cytohistologic altera‐ tions are becoming less frequent and/or fruitful.

nohistochemical positivity vary among different studies, as presented in Table 2. In the latter, studies published in the last ten years and including more than 100 cases of squamous intraepithelial lesions in histopathologic specimens are summarized [71-85], and the reported percentages of p16 immunopositivity are presented, together with the criteria and the antibodies used by the authors. As shown in the Table, different criteria have been used for p16 immunoreactivity evaluation, with some authors focusing only on diffuse immunoposi‐ tivity, some reporting any type of immunostaining, and others reporting nuclear and cyto‐ plasmic staining separately. Importantly, some authors interpret focal positivity as a falsepositive reaction. Positivity in the studies presented below varied from 5.6% to 100% for LSIL and from 45.2% to 100% for HSIL (Table 2). The percentage of immunopositivity observed in

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In one recent study [17] the two basic patterns of immunoreactivity, that is focal and diffuse, were further subdivided into groups as following: Focal positivity was subdivided into cases with occasional positive cells, dispersed or in small groups, observed either (a) mainly in the lower epithelial layers, or (b) above the basal/parabasal layer. Diffuse positivity in the horizontal plane involved either (a) all epithelial layers, or (b) only the basal, parabasal and intermediate layers, without extending to the upper third of the epithelium. In HSIL only diffuse positivity was encountered, observed in 24/25 cases (96%) (Figure 3a). In LSIL 41/55 cases (74.5%) showed some type of positivity, most commonly focal/sporadic (Figure 3b). Interestingly, a difference in HPV type distribution was observed between the two patterns of sporadic/focal positivity, involving lower vs intermediate/upper epithelial layers, and probably reflecting an earlier sporadic expression of E7 in certain lesions [17]. LSILs associated with high-risk or probable high-risk HPV types showed positivity for p16 in 25/35 cases (71.4%). Study of the pertinent literature revealed that a percentage of LSILs testing positive for HR-HPV by PCR or HC2 does not exhibit any p16 immunopositivity. Indeed, the percent‐ age of p16 positivity reported for HR-HPV positive LSILs varied from 32.4% to 94.4% [17,78,86]. Furthermore, strong p16 positivity in LSIL cases appeared to be independent of

Additionally, as shown in the Table, in several studies often appears a small group of HSILs that do not show any p16 immunoreactivity. The above observations lead to the conclusion that a negative or equivocal p16 immunostain should be carefully evaluated in conjunction with the histopathologic findings and should not be used as the main criterion for diagnosis.

In three large series of the literature reporting more than 200 cases (SILs and controls) each [71,79,81], as summarized in [17], sensitivity of p16 for the detection of SIL varied from 76.6% to 94.8%, with a value of 83.7% calculated in the total number of their cases, while specific‐ ity varied from 77.1% to 92.1%, with a value of 84.6% calculated for the total number of cases. In the same studies the positive predictive value varied from 75.7% to 94.1%. In the study by Kostopoulou et al. [17] sensitivity of p16 immunopositivity for the detection of SIL was 81.2% and specificity 85%, while the positive predictive value was 95.6%. In the study by van Niekerk et al. sensitivity and specificity for HSIL were estimated at 90% and 85%,

negative for SIL (NS) epithelia also varied between 0% and 32.7%.

HPV punctate signal pattern by ISH, in a study by Kalof et al [86].

respectively.

In recent years immunohistochemistry (IHC) is an important adjunct in many diagnoses of surgical pathology, since it may reveal certain characteristics of cells and tissues, which cannot be evaluated by morphology alone. Cervical biopsies are not an exception. Molecular studies have revealed markers that might be of utility in the diagnosis of squamous and glandular intraepithelial alterations, including cellular proteins targeted directly by viral oncoproteins, and markers related to the cell cycle, which is disturbed by multiple actions of the virus, as summarized in the above paragraphs.

#### **4.1. Immunohistochemical stains in the diagnosis of squamous intraepithelial lesions (SILs)**

The immunohistochemical stains that are currently in use in several laboratories worldwide, as well as some new promising markers are presented in the following paragraphs. The terms low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL) will be used interchangeably with CIN1 and CIN2/3, respectively.

#### *4.1.1. p16*

One extensively studied marker is p16 INK4A (hereafter referred to as p16), a cyclin-dependent kinase inhibitor, which decelerates the cell cycle and functions as a tumor suppressor, while having a role in cellular senescence. p16 affects pRb-mediated regulation of the G1/S transition [65-68]. The expression of p16 is altered in several human tumors by deletions, mutations, or methylation. Germ-line mutation carriers are predisposed to a high risk of pancreatic and breast cancers [69].

Increased expression of p16 is often observed in HPV-related intraepithelial lesions and this is mainly attributed to the presence of a feedback loop, which depends on the status of retinoblastoma protein (pRb) and the potential of high-risk HPV E7 protein to inactivate the latter [48,65,70]. Thus, it could be regarded as a marker of E7 activity. Despite the presence of high levels of p16 in SILs, its suppressor function is not normally exerted.

Several groups of investigators have examined immunohistochemically the expression of p16 in cervical squamous intraepithelial lesions and its possible correlations with lesion grade, HPV types and/or lesion "progression" (reviewed by Kostopoulou et al. [17]). Indeed, p16 is one of the best studied markers in gynaecologic pathology. However, percentages of immu‐ nohistochemical positivity vary among different studies, as presented in Table 2. In the latter, studies published in the last ten years and including more than 100 cases of squamous intraepithelial lesions in histopathologic specimens are summarized [71-85], and the reported percentages of p16 immunopositivity are presented, together with the criteria and the antibodies used by the authors. As shown in the Table, different criteria have been used for p16 immunoreactivity evaluation, with some authors focusing only on diffuse immunoposi‐ tivity, some reporting any type of immunostaining, and others reporting nuclear and cyto‐ plasmic staining separately. Importantly, some authors interpret focal positivity as a falsepositive reaction. Positivity in the studies presented below varied from 5.6% to 100% for LSIL and from 45.2% to 100% for HSIL (Table 2). The percentage of immunopositivity observed in negative for SIL (NS) epithelia also varied between 0% and 32.7%.

entering the differential diagnosis, and, c) if the diagnosis of an intraepithelial lesion is confirmed, distinguishing a low-grade from a high-grade alteration. Histopathological diagnosis of intraepithelial lesions is based on well-defined criteria, as discussed above. However, in certain cases distinguishing both low- and high-grade lesions from their mimics may pose problems [14,61], even to experienced gynecologic pathologists. Florid reactive changes and metaplastic patterns may present histopathologic features, which may cause difficulties in the distinction from HPV-induced alterations. Attempts have been made to redefine the traditional criteria for lesion diagnosis, while other efforts aimed at the adoption of new, more objective methods, which might support the former [22,23,62-64]. However, studies attempting to correlate HPV presence and replication to certain cytohistologic altera‐

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

In recent years immunohistochemistry (IHC) is an important adjunct in many diagnoses of surgical pathology, since it may reveal certain characteristics of cells and tissues, which cannot be evaluated by morphology alone. Cervical biopsies are not an exception. Molecular studies have revealed markers that might be of utility in the diagnosis of squamous and glandular intraepithelial alterations, including cellular proteins targeted directly by viral oncoproteins, and markers related to the cell cycle, which is disturbed by multiple actions of the virus, as

**4.1. Immunohistochemical stains in the diagnosis of squamous intraepithelial lesions (SILs)**

The immunohistochemical stains that are currently in use in several laboratories worldwide, as well as some new promising markers are presented in the following paragraphs. The terms low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial

One extensively studied marker is p16 INK4A (hereafter referred to as p16), a cyclin-dependent kinase inhibitor, which decelerates the cell cycle and functions as a tumor suppressor, while having a role in cellular senescence. p16 affects pRb-mediated regulation of the G1/S transition [65-68]. The expression of p16 is altered in several human tumors by deletions, mutations, or methylation. Germ-line mutation carriers are predisposed to a high risk of pancreatic and

Increased expression of p16 is often observed in HPV-related intraepithelial lesions and this is mainly attributed to the presence of a feedback loop, which depends on the status of retinoblastoma protein (pRb) and the potential of high-risk HPV E7 protein to inactivate the latter [48,65,70]. Thus, it could be regarded as a marker of E7 activity. Despite the presence of

Several groups of investigators have examined immunohistochemically the expression of p16 in cervical squamous intraepithelial lesions and its possible correlations with lesion grade, HPV types and/or lesion "progression" (reviewed by Kostopoulou et al. [17]). Indeed, p16 is one of the best studied markers in gynaecologic pathology. However, percentages of immu‐

lesion (HSIL) will be used interchangeably with CIN1 and CIN2/3, respectively.

high levels of p16 in SILs, its suppressor function is not normally exerted.

tions are becoming less frequent and/or fruitful.

summarized in the above paragraphs.

*4.1.1. p16*

breast cancers [69].

In one recent study [17] the two basic patterns of immunoreactivity, that is focal and diffuse, were further subdivided into groups as following: Focal positivity was subdivided into cases with occasional positive cells, dispersed or in small groups, observed either (a) mainly in the lower epithelial layers, or (b) above the basal/parabasal layer. Diffuse positivity in the horizontal plane involved either (a) all epithelial layers, or (b) only the basal, parabasal and intermediate layers, without extending to the upper third of the epithelium. In HSIL only diffuse positivity was encountered, observed in 24/25 cases (96%) (Figure 3a). In LSIL 41/55 cases (74.5%) showed some type of positivity, most commonly focal/sporadic (Figure 3b). Interestingly, a difference in HPV type distribution was observed between the two patterns of sporadic/focal positivity, involving lower vs intermediate/upper epithelial layers, and probably reflecting an earlier sporadic expression of E7 in certain lesions [17]. LSILs associated with high-risk or probable high-risk HPV types showed positivity for p16 in 25/35 cases (71.4%). Study of the pertinent literature revealed that a percentage of LSILs testing positive for HR-HPV by PCR or HC2 does not exhibit any p16 immunopositivity. Indeed, the percent‐ age of p16 positivity reported for HR-HPV positive LSILs varied from 32.4% to 94.4% [17,78,86]. Furthermore, strong p16 positivity in LSIL cases appeared to be independent of HPV punctate signal pattern by ISH, in a study by Kalof et al [86].

Additionally, as shown in the Table, in several studies often appears a small group of HSILs that do not show any p16 immunoreactivity. The above observations lead to the conclusion that a negative or equivocal p16 immunostain should be carefully evaluated in conjunction with the histopathologic findings and should not be used as the main criterion for diagnosis.

In three large series of the literature reporting more than 200 cases (SILs and controls) each [71,79,81], as summarized in [17], sensitivity of p16 for the detection of SIL varied from 76.6% to 94.8%, with a value of 83.7% calculated in the total number of their cases, while specific‐ ity varied from 77.1% to 92.1%, with a value of 84.6% calculated for the total number of cases. In the same studies the positive predictive value varied from 75.7% to 94.1%. In the study by Kostopoulou et al. [17] sensitivity of p16 immunopositivity for the detection of SIL was 81.2% and specificity 85%, while the positive predictive value was 95.6%. In the study by van Niekerk et al. sensitivity and specificity for HSIL were estimated at 90% and 85%, respectively.

In conclusion, the results of the above mentioned studies point towards the use of p16 immunostain in conjunction with histopathologic evaluation, since the latter remains the "gold standard" in lesion diagnosis. Addition of a consecutive p16-stained slide to the hematoxylin-eosin (HE) stained slides has been shown to improve significantly interobserv‐ er agreement for both punch and cone biopsies [18,83,87]. It is of note that a high level of agreement has been observed among pathologists in calling a staining pattern diffusely positive [18]. Moreover, p16 immunostaining may help in the identification of occult lesions [88] and of unusual high-grade lesions not easily recognized in hematoxylin-eosin stained slides [89]. The differential diagnosis from non-neoplastic alterations can be facilitated, especially in conjunction with other immunostains, as presented below. Lesion grading can be more accurate, especially concerning aggressive-appearing low-grade lesions, which could easily be upgraded [83]. p16 IHC may also be of use in evaluating cauterized cervicalresection margins, since the positive staining pattern of HGSIL is not affected by diathermy in LLETZ biopsies [76]. Awareness of the different patterns of immunoreactivity and its limitations might allow for a most proper use in certain clinicopathological settings. However, signifi‐ cant variability remains in the reported percentage of cases that stain positively for p16, and the need for standardization of sample preparation and evaluation protocols cannot be

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overemphasized [90].

(a) (b)

**Figure 3.** a – b). Diffuse p16 positivity in a HSIL (a) and focal positivity in a LSIL (b).

In the recent consensus recommendations of the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology [31], p16 was considered as the only biomarker with sufficient evidence on which to make recommendations regarding use in squamous lesions of the lower anogenital tract in the context of HPV biology. p16 immuno‐ histochemical evaluation was recommended for the distinction of high grade lesions from their mimics, as well as for diagnosis clarification of a lesion falling in the CIN2 group. However, p16 IHC is not indicated for the distinction of low grade lesions from non-HPV induced alterations or as a routine adjunct to histologic assessment of biopsy specimens with HSIL/ CIN3 or LSIL morphology. An exception to the latter concerns cases interpreted as ≤LSIL that are at high risk for missed high-grade disease, in relation to previous cytology results or HPV-


LSIL: low-grade squamous intraepithelial lesion; HSIL: high-grade squamous intraepithelial lesion; NS: negative for SIL; N: nuclear; C: cytoplasmic; ND: no data

a Only studies including more than 100 cases of squamous intraepithelial lesions in histopathologic specimens are presented.

**Table 2.** Percentages of immunopositivity for p16 in studies reported in the last ten years<sup>a</sup>

In conclusion, the results of the above mentioned studies point towards the use of p16 immunostain in conjunction with histopathologic evaluation, since the latter remains the "gold standard" in lesion diagnosis. Addition of a consecutive p16-stained slide to the hematoxylin-eosin (HE) stained slides has been shown to improve significantly interobserv‐ er agreement for both punch and cone biopsies [18,83,87]. It is of note that a high level of agreement has been observed among pathologists in calling a staining pattern diffusely positive [18]. Moreover, p16 immunostaining may help in the identification of occult lesions [88] and of unusual high-grade lesions not easily recognized in hematoxylin-eosin stained slides [89]. The differential diagnosis from non-neoplastic alterations can be facilitated, especially in conjunction with other immunostains, as presented below. Lesion grading can be more accurate, especially concerning aggressive-appearing low-grade lesions, which could easily be upgraded [83]. p16 IHC may also be of use in evaluating cauterized cervicalresection margins, since the positive staining pattern of HGSIL is not affected by diathermy in LLETZ biopsies [76]. Awareness of the different patterns of immunoreactivity and its limitations might allow for a most proper use in certain clinicopathological settings. However, signifi‐ cant variability remains in the reported percentage of cases that stain positively for p16, and the need for standardization of sample preparation and evaluation protocols cannot be overemphasized [90].

**Figure 3.** a – b). Diffuse p16 positivity in a HSIL (a) and focal positivity in a LSIL (b).

**Reference LSIL HSIL NS Evaluation Antibody**

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

[72] 35% 81.2% 0% N and/or C Polyclonal

[74] 37.2% 45.2% 3.2% N and/or C E6H4

[76] 74.1% 96.1% 7.0% N and/or C JC8

[77] 100% 98.7% 0% N or C p16

[78] 24.5% 87.5% 0% Moderate and

[80] 71.4% 100% 6% Continuous basal and

[82] 50% 96.2% 0% C and N CINtec p16 Kit

[85] 44% 99% 11% Lower ¼ of epithelium E6H4

**Table 2.** Percentages of immunopositivity for p16 in studies reported in the last ten years<sup>a</sup>

LSIL: low-grade squamous intraepithelial lesion; HSIL: high-grade squamous intraepithelial lesion; NS: negative for SIL;

Only studies including more than 100 cases of squamous intraepithelial lesions in histopathologic specimens are

[79] 90.9% 100% 7.9% C and N

[81] 57.1% 96.9% 22.9% N and C

[83] 5.6% 96.7% ND Diffuse, >1/3

[84] 26.7% 79.7% 0% N and C

N: nuclear; C: cytoplasmic; ND: no data

a

presented.

≥5% cells

≥5% cells in lower third

reactivity

strong

≥5% cells

parabasal

≥5% cells in each layer

of epithelium

≥5% cells

E6H4 (MTM)

(Abcam)

(MTM)

E6H4 (MTM)

CINtec p16 Kit (DakoCytomation)

(Biocare Medical)

(Pharmingen)

Ab7 16PO7 (Neomarkers)

(Dako)

E6H4

(Dako)

p16

Ab-4, 16P04 (Lab Vision)

(NeoMarkers)

(DakoCytomation)

p16 Histology Kit

(DakoCytomation)

E6H4 (MTM)

[71] 56.6% 84.5% 11.5% N and C

[73] 74.7 100% ND N and C

[75] 72% 94.7% 32.7% Any

In the recent consensus recommendations of the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology [31], p16 was considered as the only biomarker with sufficient evidence on which to make recommendations regarding use in squamous lesions of the lower anogenital tract in the context of HPV biology. p16 immuno‐ histochemical evaluation was recommended for the distinction of high grade lesions from their mimics, as well as for diagnosis clarification of a lesion falling in the CIN2 group. However, p16 IHC is not indicated for the distinction of low grade lesions from non-HPV induced alterations or as a routine adjunct to histologic assessment of biopsy specimens with HSIL/ CIN3 or LSIL morphology. An exception to the latter concerns cases interpreted as ≤LSIL that are at high risk for missed high-grade disease, in relation to previous cytology results or HPV- test [31]. The estimated magnitude of p16 IHC utilization, according to these recommenda‐ tions, is for <25% of cervical biopsy specimens.

nopositivity was seen in 52 of 55 cases with HPV infection detected by PCR, whereas it was seen in only 5 cases without PCR-proven HPV infection. In 4 of the latter cases, however, p16 immunoreactivity was observed, suggesting that HPV could be present though not detected

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The pattern of immunoreactivity observed in low-grade lesions and AUS cases could be perceived as cytoplasmic accumulation or retention of cyclin B1 in suprabasal squamous cells, which might reflect early events in the inhibition of G2-to-M transition, a well-known phe‐ nomenon during HPV infection in vitro. The possibility was suggested that these cyclin B1 positive cells could be viewed as a type of ''prekoilocytes", whose eventual progression to koilocytes would depend on several parameters related to the intricacies of HPV infection.

In conclusion, cyclin B1 positivity above the basal/parabasal layers correlates significantly with HPV detection and could be a marker of HPV presence. Thus, it might constitute a helpful finding in difficult to diagnose cases. Immunopositivity in a specimen showing non-diagnostic atypia should prompt reevaluation and/or HPV testing, as it is likely that the case could represent a genuine low-grade intraepithelial lesion. Thus, a search for "pre-koilocytes" with

**Figure 4.** Cyclin B1 immunopositivity above the basal/parabasal cells correlates significantly with HPV detection.

invasive cervical carcinomas, although the exact mechanisms are not clear [16].

**Cyclin E.** Cyclin E is another important cell cycle regulator, promoting G1 transition, which has been reported to exhibit increased expression in squamous intraepithelial lesions and

Moderate to strong immunopositivity for cyclin E was observed in 92.6% and in 91.6% of LSIL and HSIL, respectively, in a study by Keating et al., being positive in 38/41 HR-HPV positive cases. In a group of nondiagnostic squamous atypias cyclin E positivity was associated with HPVpositivity[16].InastudybyBahnassyetal.cyclinEstainingincreasedfromCIN1toinvasive carcinoma (from 16.7% to 88.4%, respectively), while gene amplification was detected in 11.1% of CIN1 cases, in 45.5% of CINII, in 55.3% of CINIII, and in 88.4% of carcinoma cases [94].

B1 immunostaining might be useful in certain cases.

by PCR.

Finally, another aspect of p16 immunostaining is the possibility of correlation with lesion "progression". It has been suggested that certain phases of a given HR-HPV-associated neoplastic process may have different indices of p16 expression [16]. Although the detailed examination of this subject is not included in the aim of the present text, it should be mentioned that, in an interesting study by Hariri and Oster, 25/26 low-grade lesions with negative p16 staining (concerning diffuse staining) and a minimum follow-up period of five years had a benign or normal outcome, revealing a negative predictive value of p16 in predicting the outcome of CIN 1 cases as high as 96% [80]. In a study including conization specimens with coexisting CIN1 and CIN3 areas, all CIN1 were p16 positive [91], while p16 staining did not predict persistence or clearance of HR-HPV after treatment for CIN in a study by Branca et al. [72]. In a recent report by Ozaki et al. [85] including 99 patients with CIN1 and follow-up data, 26/76 cases in the regression group showed p16 immunopositivity vs 17/23 in the persistence/ progression groups.

#### *4.1.2. Cyclins*

Cyclins along with cyclin-dependent kinases and their inhibitors are important molecules for the orderly progression of cells through phases of the cell cycle. Their immunohistochemical evaluation has been reported to be of help in the diagnosis of SIL in cervical specimens. Cyclin E is uncommonly expressed in epithelia not infected by HPV and its conspicuous immuno‐ positivity may facilitate the recognition of SIL [16]. In addition, cyclin B1 immunoreactivity above the basal/parabasal cells correlates significantly with HPV detection and could be a marker of HPV presence [21]. Cyclins D and A have been also studied as possible markers of HPV-related lesions.

**Cyclin B1.** It has been reported that E6/E7 oncoproteins of HPV type 18 induced changes in the expression of cell cycle regulatory proteins before immortalization [92]. Significantly increased expression was noted for cyclin B and its transcriptional activation was documented. In 2000, Southern et al. demonstrated increased cyclin B1 expression in HGSILs [93]. In their study cyclin B protein was up-regulated and persisted into the upper epithelial layers in parallel with cyclin A expression in high-grade squamous intraepithelial lesions.

In a study performed in our laboratory, cyclin B1 immunopositivity above the basal/parabasal cells correlated significantly with HPV detection (p<0.001) (Figure 4). In all cases of HSIL immunopositivity for cyclin B1 extended above the basal/parabasal layers, most often involving the superficial layers as well [21]. Furthermore, increased cyclin B1 immunoreac‐ tivity was observed in 51/52 low-grade lesions (98.07%), and in seven of 15 biopsies (46.6%) characterized as atypia of unknown significance (AUS). Six of these seven cases tested HPVpositive by PCR.

The main staining pattern observed in low-grade lesions and non-diagnostic atypias in the above study consisted of sporadic cyclin B1 staining in mature squamous polygonal cells often just above the basal layers, with slight differences between flat and elevated lesions. Immu‐ nopositivity was seen in 52 of 55 cases with HPV infection detected by PCR, whereas it was seen in only 5 cases without PCR-proven HPV infection. In 4 of the latter cases, however, p16 immunoreactivity was observed, suggesting that HPV could be present though not detected by PCR.

test [31]. The estimated magnitude of p16 IHC utilization, according to these recommenda‐

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

Finally, another aspect of p16 immunostaining is the possibility of correlation with lesion "progression". It has been suggested that certain phases of a given HR-HPV-associated neoplastic process may have different indices of p16 expression [16]. Although the detailed examination of this subject is not included in the aim of the present text, it should be mentioned that, in an interesting study by Hariri and Oster, 25/26 low-grade lesions with negative p16 staining (concerning diffuse staining) and a minimum follow-up period of five years had a benign or normal outcome, revealing a negative predictive value of p16 in predicting the outcome of CIN 1 cases as high as 96% [80]. In a study including conization specimens with coexisting CIN1 and CIN3 areas, all CIN1 were p16 positive [91], while p16 staining did not predict persistence or clearance of HR-HPV after treatment for CIN in a study by Branca et al. [72]. In a recent report by Ozaki et al. [85] including 99 patients with CIN1 and follow-up data, 26/76 cases in the regression group showed p16 immunopositivity vs 17/23 in the persistence/

Cyclins along with cyclin-dependent kinases and their inhibitors are important molecules for the orderly progression of cells through phases of the cell cycle. Their immunohistochemical evaluation has been reported to be of help in the diagnosis of SIL in cervical specimens. Cyclin E is uncommonly expressed in epithelia not infected by HPV and its conspicuous immuno‐ positivity may facilitate the recognition of SIL [16]. In addition, cyclin B1 immunoreactivity above the basal/parabasal cells correlates significantly with HPV detection and could be a marker of HPV presence [21]. Cyclins D and A have been also studied as possible markers of

**Cyclin B1.** It has been reported that E6/E7 oncoproteins of HPV type 18 induced changes in the expression of cell cycle regulatory proteins before immortalization [92]. Significantly increased expression was noted for cyclin B and its transcriptional activation was documented. In 2000, Southern et al. demonstrated increased cyclin B1 expression in HGSILs [93]. In their study cyclin B protein was up-regulated and persisted into the upper epithelial layers in

In a study performed in our laboratory, cyclin B1 immunopositivity above the basal/parabasal cells correlated significantly with HPV detection (p<0.001) (Figure 4). In all cases of HSIL immunopositivity for cyclin B1 extended above the basal/parabasal layers, most often involving the superficial layers as well [21]. Furthermore, increased cyclin B1 immunoreac‐ tivity was observed in 51/52 low-grade lesions (98.07%), and in seven of 15 biopsies (46.6%) characterized as atypia of unknown significance (AUS). Six of these seven cases tested HPV-

The main staining pattern observed in low-grade lesions and non-diagnostic atypias in the above study consisted of sporadic cyclin B1 staining in mature squamous polygonal cells often just above the basal layers, with slight differences between flat and elevated lesions. Immu‐

parallel with cyclin A expression in high-grade squamous intraepithelial lesions.

tions, is for <25% of cervical biopsy specimens.

progression groups.

HPV-related lesions.

positive by PCR.

*4.1.2. Cyclins*

The pattern of immunoreactivity observed in low-grade lesions and AUS cases could be perceived as cytoplasmic accumulation or retention of cyclin B1 in suprabasal squamous cells, which might reflect early events in the inhibition of G2-to-M transition, a well-known phe‐ nomenon during HPV infection in vitro. The possibility was suggested that these cyclin B1 positive cells could be viewed as a type of ''prekoilocytes", whose eventual progression to koilocytes would depend on several parameters related to the intricacies of HPV infection.

In conclusion, cyclin B1 positivity above the basal/parabasal layers correlates significantly with HPV detection and could be a marker of HPV presence. Thus, it might constitute a helpful finding in difficult to diagnose cases. Immunopositivity in a specimen showing non-diagnostic atypia should prompt reevaluation and/or HPV testing, as it is likely that the case could represent a genuine low-grade intraepithelial lesion. Thus, a search for "pre-koilocytes" with B1 immunostaining might be useful in certain cases.

**Figure 4.** Cyclin B1 immunopositivity above the basal/parabasal cells correlates significantly with HPV detection.

**Cyclin E.** Cyclin E is another important cell cycle regulator, promoting G1 transition, which has been reported to exhibit increased expression in squamous intraepithelial lesions and invasive cervical carcinomas, although the exact mechanisms are not clear [16].

Moderate to strong immunopositivity for cyclin E was observed in 92.6% and in 91.6% of LSIL and HSIL, respectively, in a study by Keating et al., being positive in 38/41 HR-HPV positive cases. In a group of nondiagnostic squamous atypias cyclin E positivity was associated with HPVpositivity[16].InastudybyBahnassyetal.cyclinEstainingincreasedfromCIN1toinvasive carcinoma (from 16.7% to 88.4%, respectively), while gene amplification was detected in 11.1% of CIN1 cases, in 45.5% of CINII, in 55.3% of CINIII, and in 88.4% of carcinoma cases [94].

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.

*4.1.4. ProEx C*

tion with p16.

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‐

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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

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

reactive epithelial changes from SILs than MiB-1.

only one third of the cases required performance of the third stain.

#### *4.1.3. Ki67*

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

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

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

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