Stratification of HPV-induced cervical pathology using the virally encoded molecular marker E4 in combination with p16 or MCM

Abstract

High-risk human papillomavirus (HPV) types cause cervical lesions of varying severity, ranging from transient productive infections to high-grade neoplasia. Disease stratification requires the examination of lesional pathology, and possibly also the detection of biomarkers. P16(INK4a) and MCM are established surrogates of high-risk HPV E6/E7 activity, and can be extensively expressed in high-grade lesions. Here we have combined these two cellular biomarkers with detection of the abundant HPV-encoded E4 protein in order to identify both productive and transforming lesions. This approach has allowed us to distinguish true papillomavirus infections from similar pathologies, and has allowed us to divide the heterogeneous CIN2 category into those that are CIN1-like and express E4, and those that more closely resemble nonproductive CIN3. To achieve this, 530 lesional areas were evaluated according to standard pathology criteria and by using a multiple staining approach that allows us to superimpose biomarker patterns either singly or in combination onto an annotated hematoxylin and eosin (H&E) image. Conventional grading of neoplasia was established by review panel, and compared directly with the composite molecular pathology visualized on the same tissue section. The detection of E4 coincided with the onset of vacuolation, becoming abundant in koilocytes as the MCM marker declined and cells lost their defined nuclear margins as visualized by standard H&E staining. Of the dual marker approaches, p16(INK4a) and E4 appeared most promising, with E4 generally identifying areas of low-grade disease even when p16(INK4a) was present. Extensive p16(INK4a) expression usually coincided with an absence of E4 expression or its focal retention in sporadic cells within the lesion. Our results suggest that a straightforward molecular evaluation of HPV life-cycle deregulation in cervical neoplasia may help improve disease stratification, and that this can be achieved using complementary molecular biomarker pairs such as MCM/E4 or, more promisingly, p16(INK4a)/E4 as an adjunct to conventional pathology.

Figure 1. Overlay of biomarker patterns onto annotated Hemotoxylin and Eosin Pathology

A. Individual tissue sections were subject to immunofluorescence staining to detect the biomarkers E4 using a TVG405-Alexa 488 conjugate (green), MCM using an anti-mouse Alexa 594 secondary antibody (red) and cellular DNA using 4′,6-Diamidino-2-Phenylindole (DAPI; blue). Individual stains were recorded digitally at high resolution (lower panels) before the section was processed for p16^INK4a staining and development in 3_Amino_9_ethylcarbazole (AEC; brown). The AEC image was digitally captured, before the tissue section was cleared of the AEC substrate and stained with Carazzis (X2) Hemotoxylin and Eosin. All H&E stained images were then examined independently by three pathologists, and regions with discrete pathology phenotypes recorded. The primary categories of HPV-associated pathology comprised CIN1, CIN2 and CIN3, with the general term ‘non-CIN’ being used to encompass a variety of non HPV-associated pathologies such as inflammation and metaplasia. A ‘normal’ classification was given when there was no histological abnormality. B. The 3-colour immunofluorescence stain is shown on the left. Single channel coloured images (collected as described in A) were extracted from the immunofluorescence image or from the p16^INK4a AEC stain and were superimposed onto the H&E images (under the heading ‘Pathology overlay’). The simple dual marker molecular pathologies (i.e. not overlayed onto the H&E image) are shown on the right to reveal the relative distributions of E4/MCM and E4/ p16^INK4a (under the heading ‘Dual marker molecular pathology). The image shown is typical of those used to prepare the more detailed images of neoplasias used in subsequent figures.

Figure 2. Distribution of the HPV_E4, p16^INK4a and MCM2 biomarkers in lesions unambiguously classified as CIN1

A. Biomarker patterns typically associated with CIN1. The E4/MCM (green/red) biomarker patterns are shown in the immunofluorescence image to the left of the figure. To facilitate comparison with lesional pathology, the E4/MCM (green/red) and p16^INK4a (brown) biomarkers are overlayed onto (and shown alongside) the basic H & E stain in the central part of the figure. The E4/MCM (green/red) and E4/ p16^INK4a (green/brown) biomarker overlays are shown separately to the right of the figure so that their relative distributions can be observed. The Biomarker patterns seen in HPV associated CIN1 are described below.

1. i) Most CIN1 are productive infections, with MCM (red) reaching and extending into the E4 expressing layers (green). In this lesion, MCM and p16^INK4a have broadly similar distributions.

2. ii) Although p16^INK4a (brown) and MCM (red) expression can have different distributions, MCM again extends into the E4-positive layers (green). In this lesion, MCM extends higher into the epithelial layers than p16^INK4a.

3. iii) A small number of consensus CIN1 (6/33) failed to show E4 (green) biomarker expression. p16^INK4a (brown) expression extends into the upper epithelial layers and is more extensive than MCM (red).

B. Correlation of Biomarker patterns with Pathology. The E4/MCM (green/red) biomarker distribution in consensus CIN1 are shown as immunofluorescence images to the left of the figure, and overlayed onto H & E pathology towards the centre. The higher magnification shown on the right allows correlation of specific pathology characteristics with the accumulation of E4 and the decline of MCM.

1. i) In the majority of CIN1, the E4 (green) biomarker accumulates during the process of koilocyte formation. Arrows labelled 1 to 4 show the progressive vacuolation that accompanies E4 accumulation (beginning at arrow 2 and highest at arrow 4), and the loss of the MCM biomarker (lowest at arrow 4).

2. ii) Despite some differences in cell morphology and tissue architecture amongst CIN1, the distribution of biomarkers described above (i) is conserved.

3. iii) In one (out of 33) consensus CIN1, koilocyte-like cells we present but were devoid of markers of productive infection, including the E4 (green) biomarker and associated MCM (red) staining.

Figure 3. Relevance of the HPV_E4, p16^INK4a and MCM biomarkers in consensus CIN3 lesions

A. Biomarker Patterns Typically Associated with CIN3. The E4/MCM (green/red) and p16^INK4a (brown) biomarker images are formatted as outlined in Fig.2A. The biomarker patterns seen in HPV-associated CIN3 are described below.

1. i) In the majority of CIN3, the E4 (green) biomarker is absent, and both the MCM (red) and p16^INK4a (brown) biomarkers extend uniformly through the full thickness of the epithelium.

2. ii) Despite differences in lesion size and morphology, the biomarker patterns described above (i) were broadly similar in other CIN3.

B. Expression of the E4 biomarker is an occasional occurrence in CIN3.

1. i) In a small number of CIN3 (4/68), focal areas of E4 (green) were apparent. Both p16^INK4a (brown) and MCM (red) extended through the full thickness of the lesion with evidence of nuclear crowding as seen in Fig 3A. Panel A is shown enlarged in Fig. 3C.

2. ii) In one case (out of 68), the E4 (green) biomarker was extensive, but was confined to regions showing low-grade pathology that lacked strong p16^INK4a (brown) biomarker staining. Panel B is shown enlarged in Fig. 3B.

C. Pathology associated with E4 expression in CIN3. The three images to the left show an enlargement of Fig. 3B(i) panel A. Although vacuolated cells are sometimes apparent in CIN3 (arrows marked ‘v’), they are not necessarily associated with expression of the E4 biomarker (Fig. 2B(i)). The pathology associated with E4 expression in CIN3 is more clearly shown in the images to the right, which is an enlargement of panel B from Fig.3B(ii). This region of low-grade pathology was contained within the CIN3 area. The pattern of vacuolation and koilocyte formation is comparable to that seen in CIN1 (Fig. 2B), with E4 expression beginning at the first sign of vacuolation (arrows labelled 1) and becoming prominent as the MCM biomarker is lost (arrows labelled 2).

Figure 4. Expression of HPV_E4 along with p16^INK4a and MCM suggests two categories within the consensus CIN2 group

A. HPV_E4-positivity amongst consensus CIN2. The E4/MCM (green/red) and p16^INK4a (brown) biomarker images are formatted as outlined in Fig.2A. The biomarker patterns seen in E4-positive HPV-associated CIN2 are described below.

1. i) High cell density and the expression of MCM precedes the accumulation of E4 in a subset of cells showing evidence of vacuolation. P16^INK4a and MCM expression extend throughout the full thickness of the epithelium. Panel A is enlarged in Fig.5.

2. ii) Despite differences in epithelial thickness, the biomarker pattern is preserved in other consensus CIN2.

3. iii) As in CIN1, some CIN2 show a more extensive expression of MCM into the upper epithelial layers when compared to p16^INK4a. E4 expression is limited to cells close to the epithelial surface. Panel B is enlarged in Fig.5 to illustrate the correlation between pathology and biomarker patterns.

B. HPV_E4 negativity amongst consensus CIN2.

1. i) Loss of the E4 biomarker in a proportion of CIN2, can be associated with extensive expression of the p16^INK4a /MCM biomarkers throughout the thickness of the epithelium, as well as an absence of obvious differentiation at the level of pathology. Panel C is enlarged in Fig. 5.

2. ii) MCM can extend closer throughout the epithelium more robustly than p16^INK4a without the expression of the E4 biomarker. The panel shown in D is enlarged in Fig. 5 to show the absence of key pathology features apparent in the E4-positive CIN2.

Figure 5. Expression of the HPV_E4 biomarker in CIN2 is associated with discrete regions of CIN1-like pathology

A.

1. i) Pathology associated with E4 expression in CIN2. The six images show enlargements of the regions that are boxed in Fig. 4A(i) and (iii) (H&E images). The pattern of vacuolation and koilocyte formation is similar to that seen in CIN1 (Fig. 2B), with E4 expression, vacuolation and MCM decline coinciding closely (arrows 1&2).

B.

1. ii) The six images show an enlargement of the regions that are boxed in Fig 4B(i) and (ii). In these lesions, vacuolated cells are sometimes apparent (arrows marked ‘v’), but are not necessarily associated with expression of the E4 biomarker (Fig. 4B(i) and (ii)).

Figure 6. Division of Cervical Pathologies according to the Presence and Distribution of the Molecular Markers E4 and p16^INK4a

A. Lesional areas where there was total agreement amongst the panel of pathologists. The columns in each graph show the individual diagnostic opinions provided by the pathologist panel after review of the H&E stained slides, as either non-CIN, CIN1, CIN2 or CIN3. Graphs shown in A include only lesional areas where there was total agreement amongst the pathologist panel. This standard pathology-grading is stratified according to whether the diagnosed areas were subsequently found to be HPV-E4-positive (green edged columns/left-most graph) or E4-negative (red edged columns/right-most graph), and to what extent the p16^INK4a expression extended through the epithelium. Lesional areas showing full thickness p16 staining are indicated as dark brown columns, with lower levels of staining being shown in lighter shades of brown. Lesional areas that lacked p16^INK4a staining are indicated by white columns. In general, the patterns fit with our current model of life-cycle deregulation in high-grade neoplasia, with an absence of both markers in the ‘total-agreement’ non-CIN group. As described in the text, E4 expression in CIN3 (marked by an asterisk) was typically sporadic and in a small number of cells close to the epithelial surface. B. All lesional areas including those where there was disagreement amongst the panel of pathologists. The columns show the individual diagnostic opinions provided by the pathologist panel on all lesional areas, irrespective of whether there was agreement or disagreement between individual pathologists. Labelling is as described in ‘A’ above. Because the H&E-based diagnostic opinion often differed between the individual pathologists, the pattern of the p16^INK4a and E4 staining typical of non-CIN, CIN1, CIN2 and CIN3 (shown in Fig.6A) is less apparent, with some evidence of virus infection in the ‘proposed’ non-CIN group, and an absence of biomarker staining in some ‘proposed’CIN1.

Figure 7. Molecular Principles Underlying the use of p16, MCM and E4 as HPV-Associated Disease Biomarkers

A. In uninfected epithelium, the cellular MCM protein (red) is usually detectable at low levels only in the basal and parabasal cell layers as a result of cell-cycle stimulation by growth factors. This facilitates the phosphorylation of pRb by cyclin-dependent kinases, the release of the E2F transcription factor, and the regulated expression of MCM. During normal metaplasia or wound healing, MCM may also be detected in the upper epithelial layers. The cellular p16^INK4a protein is also stimulated by E2F, but does not usually accumulate to detectable levels in uninfected epithelium. It provides feedback-regulation on the activity of cyclin-dependent kinases. p16^INK4a is sometimes visualised as a weak cytoplasmic stain in cells undergoing senescence (pale brown). The HPV-encoded E4 protein is never expressed in uninfected epithelium and E4 antibodies show no reactivity with cellular proteins. B. (i) In HPV-infected epithelial tissue, the high-risk E6 & E7 genes (red) are expressed together from the viral early promoter (PE), and function to drive cell-cycle entry in order to allow cell proliferation and genome amplification. The high-risk E4 gene (green) is expressed from a spliced mRNA, and becomes abundant following the activation of the viral late promoter (PL) as the infected cell exits the cell cycle and commits to true differentiation. (ii) E6 and E7 are expressed at low level in the cell, but the consequences of their presence can be visualised by alterations in the presence of p16^INK4a and MCM. The association of E7 with pRb leads to E2F release irrespective of growth factor stimulation. This allows MCM and also p16^INK4a to accumulate to higher levels than are typically seen in uninfected epithelium where expression is dependent on cyclin-dependent kinase activation. The E7 protein also acts to increase the transcription of p16^INK4a as a result of epigenetic modification of the p16^INK4a promoter. In this context, p16^INK4a and MCM can be used with caution as surrogate markers of E6/E7 deregulation. The viral E4 protein becomes abundant in the upper layers of HPV-infected epithelium as a result of viral late promoter activation and the cleavage of the full-length E4 protein by calpain. Calpain-cleavage exposes a C-terminal multimerization motif in E4, which allows its assembly into amyloid-like fibres. The high-level accumulation of E4-amyloid is thought to coincide with progression of the infected cell through the G2 phase of the cell cycle and eventually to cell-cycle exit, explaining its appearance as MCM levels decline.