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Keywords:

  • human papillomavirus;
  • tissue microarray;
  • biomarker;
  • cervical neoplasia;
  • human kallikrein 7

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Overexpression of kallikrein 7, a proteolytic enzyme important for epithelial cell shedding, may be causally involved in carcinogenesis, particularly in tumor metastasis and invasion. In this study, we have evaluated hK7 (human kallikrein 7) protein levels by immunohistochemistry in 367 cervical histological samples including 35 cases of cervicitis, 31 low-grade cervical intraepithelial neoplasia, 51 high-grade cervical intraepithelial neoplasia (H-SIL), 197 squamous cervical carcinomas (SCC) and 53 cervical adenocarcinomas. We have observed that hK7 staining increased with the severity of cervical disease. Intense hK7 staining was found in 15.2% of cervicitis samples, in contrast to 55% of H-SIL and 68% of SCC. Moreover, 92.5% of adenocarcinomas also exhibited intense hK7 staining. Differences in the expression of hK7 could potentially be used as a biomarker for the characterization of different stages of cervical disease.

Cervical cancer is the second leading cancer in women worldwide. Current estimates indicate that ∼494,000 women are diagnosed with cervical cancer worldwide and almost 274,000 die from the disease every year.1, 2 It has been recognized that persistent infection with high-risk (HR) human papillomavirus (HPV) types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) is a necessary cause for the development of preinvasive and invasive cervical cancer (ICC).3 Although HPV infection has a prevalence of about 20–40% among sexually active and cytological normal women, 93–99.7% of cervical cancers are positive for HR-HPV.4, 5 There are 2 main histological types of ICC: squamous cell carcinomas (SCC) and adenocarcinomas. The incidence of SCC in developed countries has significantly decreased because of cytology screening (Pap test). However, invasive cases still occur because Pap test results may have a high variability owing to its subjective morphological criteria. Moreover, the proportion of adenocarcinomas has increased because Pap test is not as efficacious in detecting this lesion.6–8 Therefore, the identification of new biomarkers for the presence of cervical dysplasia, SCC and adenocarcinomas could improve the detection of lesions with higher risk of progression.

We have previously shown that HPV18-immortalized keratinocytes exhibit higher KLK7 transcript levels when compared with normal and HPV16-immortalized human keratinocytes.9 Recent data suggest that kallikreins may be causally involved in carcinogenesis, particularly in tumor metastasis and invasion.10–12 The protein kallikrein 7 (hK7), also known as human stratum corneum chymotryptic enzyme, is a secreted chymotrypsin-like serine protease first identified in human skin extracts.13, 14 Although it has been shown that this protein is involved in degradation of several components of the extracellular matrix, such as desmoglein-1, desmocollin-1, corneodesmossomes and fibronectin, its role in human malignancies remains unclear.15–19 High hK7 expression (messenger RNA and/or protein level) has been observed in breast20 and ovary tumors21, 22 and is considered an unfavorable prognostic marker for these pathologies.10

To date, few studies have addressed hK7 expression in cervical tumors. Using immunohistochemistry (IHC), Tian et al.23 observed a higher hK7 expression rate in cervical adenocarcinomas (80% positivity) compared with normal endocervical glands (50% positivity). On the other hand, another study reported higher hK7 expression levels in SCC (80% positivity) compared with adenocarcinomas (20% positivity). Furthermore, in this study, hK7 expression was not detected in normal cervical keratinocytes.24 In sight of these conflicting results, we assessed the expression of hK7 in a larger number of cervical specimens. In this study, we analyzed the expression of hK7 in 367 histological samples of cervical lesions of different grades. We observed that hK7 levels increased with the severity of cervical lesions, suggesting that hK7 might be a useful biomarker for high-grade cervical dysplasia and cancer.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Tissue samples and tissue microarray construction

Cervical tissue samples were obtained from the Department of Anatomic Pathology, Medical and Research Centre, Hospital A. C. Camargo, São Paulo, Brazil, the Pathology Division, Adolfo Lutz Institute, São Paulo, Brazil and the Laboratory of Medical Investigation (LIM) 14, Department of Pathology, São Paulo University School of Medicine, Brazil. Ethical approval for this study was granted by local research ethics committees. In this study, 379 cervical samples were used to construct 3 TMA (tissue microarray) platforms. Twelve samples were inadequate for analysis and the remaining 367 corresponding to 35 cases of cervicitis, 31 low-grade squamous intraepithelial lesion (L-SIL), 51 high-grade squamous intraepithelial neoplasia (H-SIL), 197 SCC and 53 cervical adenocarcinomas were analyzed. Samples from cervicitis, L-SIL and H-SIL were spotted in duplicate.

Previously fixed and paraffin embedded tissues were first sectioned in 3-μm slices, which were hematoxylin-eosin stained for selection of the appropriated tissue area. The correspondent selected areas of each tissue sample were then collected from the donor-embedded tissues with a 1-mm gauge cylinder and transferred to a receptor block using a manual tissue microarrayer (Beecher Instruments, Sun Prairie, WI). TMA 3-μm sections were cut from the receptor block and processed as described later.

Immunohistochemical hK7 detection

After deparaffinization in xylene and rehydration in alcohol, antigen retrieval was performed by incubation in citrate buffer pH 6.0 in a pressure cooker for 3–4 min. Endogenous peroxidase activity was inactivated by incubation with 3% hydrogen peroxide. Nonspecific avidin binding was also blocked (DAKO, X0590, Glostrup, Denmark). The hK7 antibody used in this study was kindly provided by Dr. Eleftherios Diamandis, Mount Sinai Hospital, Toronto, Canada. This polyclonal antibody was generated by immunization of rabbits with the full-length prohuman hK7 protein (clone 493-13), derived from HEK293 (Eleftherios Diamandis, personal communication). Samples were incubated with anti-hK7 (1:1,000) in 1% bovine serum albumin-phosphate buffered solution for 8–18 hr at 4°C. The slides were then incubated for 30 min at 37°C with secondary biotinylated goat anti-mouse/rabbit Ig, followed by incubation for 30 min at 37°C with streptavidin and biotinylated peroxidase (StreptABComplex/HRP Duet Mouse/Rabbit DakoCytomation, K0492, Glostrup, Denmark). Slides were developed using 3,3′-diaminobenzidine tetrahydrochloride (Sigma, D-5637, USA), 6% H2O2 and dimethyl sulphoxide and counterstained with Harris' hematoxylin. Sections derived from an ovarian papillary serous adenocarcinoma were used as positive control and were incubated in the absence or in the presence of anti-hK7 antibody. Sections of normal cervical tissues were used as negative control.

Evaluation of tissue staining

The positive immunohistochemical reaction was evaluated considering the percentage of stained cells in each representative histopathological category (cervicitis, L-SIL, H-SIL, SCC and adenocarcinomas). The immunohistochemical reactions were performed in duplicate on the cuts obtained from the TMA blocks. Only cores with more than 25% of cervical epithelium tissue were analyzed. The evaluation was performed by examining the epithelium of the whole fragment of each sample present in the TMA platforms under magnification of 200×. Positive reactions were semiquantitatively scored as follows: “negative/weak” (<10% stained cells), “moderate” (10–50% stained cells) and “intense” (>50% stained cells). Immunohistochemical evaluation was performed blindly by 2 independent observers (ALF and FAS); the very few discordant results were discussed by both observers and a final score was established.

Statistical analysis

Comparisons among proportions were done using the Fisher exact test, and the linear-by-linear association test was used to evaluate trends among proportions. Odds ratios (ORs) and 95% confidence intervals were estimated using 2 × 2 contingency tables. All statistical analyses were done with the Statistical Package for the Social Sciences 15.0 software. Statistical significance was based on a value of p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Overall, the TMA platforms used in this study were representative of different cervical pathologies. An evaluation with hematoxylin-eosin staining revealed adequate results for most specimens. Only cores with more than 25% of cervical epithelium tissue were analyzed. Unfortunately, such tissues were not amenable for amplification of HPV genomes (or housekeeping genes, such as globin). Therefore, no data on HPV status are available in this study.

hK7 expression was detected by IHC, and staining was initially determined as negative/weak, moderate and intense according to the percentage of cells positive for this protein (0–10%, 10–50% and >50% stained cells, respectively). We have analyzed hK7 expression in 367 cervical samples, namely 35 (9.5%) cervicitis, 31 (8.4%) L-SIL, 51 (13.9%) H-SIL, 197 (53.7%) SCC and 53 (14.5%) adenocarcinomas. We observed that hK7 staining frequency in cervicitis samples was very similar to exhibited by L-SIL samples (Supporting Information Fig. 1). This observation is consistent to the fact that cervicitis and L-SIL have similar characteristics of the cytopathogenic effects of a replicative HPV infection and tend to regress spontaneously.25, 26 Therefore, samples diagnosed as cervicitis and L-SIL were grouped in a single histopathological cathegory (Cerv + L-SIL) and used as the reference category for comparisons with H-SIL, SCC and adenocarcinomas.

Moreover, as we did not observe significant differences between the percentage of negative/weak and moderate stained samples within each histopathological category (Supporting Information Fig. 2), we grouped them in a single category (<50% stained cells). We then analyzed the staining intensity in 2 categories: <50% and >50% of stained cells. Using these criteria, we observed a clear pattern where the percentage of samples exhibiting intense staining for hK7 increased from reference category (15.2%) to H-SIL (55%) and SCC (68%). Furthermore, when we analyzed adenocarcinomas, we observed that 92.5% of the specimens exhibited more than 50% of positive cell staining for hK7. The proportion of samples expressing high levels of hK7 between the reference and all other histological categories was statistically different (p < 0.001) (Fig. 1).

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Figure 1. Distribution of hK7 staining according to diagnostic classification. Samples were grouped depending on the staining intensity as negative, weak or moderate (<50% positivity) and intense (>50% positivity). Cerv + L-SIL: cervicitis and low-grade squamous intraepithelial lesions; H-SIL: high-grade squamous intraepithelial lesions; SCC: squamous cervical carcinomas; ADENO: cervical adenocarcinomas. Numbers on top of each bar correspond to the number of samples in each category. *p-values.

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Finally, we observed a significant positive association between high hK7 expression level (>50% stained cells) and the grade of cervical lesion. Intense positivity for hK7 (>50% stained cells) was significantly associated with H-SIL (OR = 6.82, CI: 2.8–16.2) and SCC (OR = 11.9, CI: 5.7–24.8), when compared with cervicitis and L-SIL samples group. The trend observed between hK7 positivity and disease severity was also statistically significant (p < 0.001). Importantly, the association with hK7 positivity was particularly stronger with adenocarcinomas (OR = 68.6, CI: 20.2–232.6) (Table 1).

Table 1. Association of intense hK7 positivity and grade of cervical lesion
inline image

Figure 2 shows a representative immunostaining for hK7 in cervical samples. We observed that in the tissue corresponding to L-SIL less than 50% of cells were positive for hK7 (Fig. 2a). Interestingly, a subset of the cervicitis and L-SIL samples that were positive for hK7 exhibited nuclear, cytoplasmic and/or membrane staining (data not shown). On the other hand, H-SIL, SCC and adenocarcinoma samples displayed a strong cytoplasmic hK7 staining throughout the lesion, whereas no staining was observed in stromal cells (S) (Figs. 2b2d, respectively). Normal glandular epithelium did not present significant staining (Fig. 2b) as well as normal squamous epithelium (data not shown). The weak staining observed in normal glandular epithelium (GL) (Fig. 2b) is considered nonspecific staining of mucus present in the endocervical lumen. This is highlighted in the inset of Figure 2d, which shows a typical hK7 staining of normal endocervical tissue.

thumbnail image

Figure 2. Immunohistochemical analysis of hK7 expression in cervical samples. Representative immunoreactivity of hK7 in (a) low-grade squamous intraepithelial lesion (L-SIL), (b) high-grade squamous intraepithelial lesion (H-SIL) and normal glandular epithelium (GL), (c) squamous cervical carcinoma (SCC) and (d) adenocarcinoma (ADENO). Less than 50% of stained cells were observed in L-SIL, whereas H-SIL, SCC and adenocarcinoma samples displayed cytoplasmic staining throughout the lesion. L-SIL: low-grade squamous intraepithelial lesion; SCC: squamous cervical carcinoma; ADENO: adenocarcinoma; GL: normal glandular epithelium; S: stromal cells; L: lumen. Samples incubated in the absence of primary antibody exhibited no immunostaining [inset in (c)]. Normal glandular epithelium [inset in (d)]. Magnification: a, b and c, ×100; d, ×400.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Kallikreins are a subgroup of serine proteases with several roles in normal physiological functions (e.g., digestion, coagulation and cellular and humoral immunity) and in the pathology of several diseases (e.g., cancer and neurodegenerative disorders).10, 27–31 Several reports describe that hK7 plays an important role in the normal physiology of the skin, particularly in epidermal homeostasis,13, 14 by degrading intra- and extracellular components of the epidermis important for cellular cohesiveness and facilitating cell desquamation during the terminal stages of normal epidermal turnover.10, 15–19 On the other hand, deregulated expression of hK7 is involved in skin pathologies, such psoriasis,32 abnormal keratinization33 and inflammatory reactions, because of its ability to activate proinflammatory cytokines, such as interleukin-1β (IL-1 β).34, 35 Almost 15 kallikrein types have been identified, and nowadays the tissue kallikreins are considered a rich source of cancer biomarkers.11, 36, 37

In this study, we aimed to examine by IHC the expression of hK7 in a large set of cervical samples, including cervicitis, L-SIL, H-SIL, SCC and adenocarcinomas. We show that hK7 expression is upregulated in high-grade cervical lesions, squamous cell carcinomas and adenocarcinomas of the cervix when compared with L-SIL and cervicitis. Although a very low proportion (15.2%) of cervicitis and L-SIL exhibited intense positivity for hK7, this frequency increased significantly in H-SIL (55%) and SCC (68%). Therefore, we observed a significant trend between hK7 positivity and severity of cervical disease. Once biological aggressiveness of neoplastic cells is related to their ability to proliferate abnormally, invade normal tissues and metastatize, the increased proteolytic degradation of the extracellular matrix could be directly associated to hK7 overexpression in cervical cancer. A study evaluating the association between hK7 expression and risk of cervical disease progression is warranted.

The percentage of SCC and adenocarcinomas presenting moderate or intense positivity for hK7 is in agreement with previous reports.23, 24 On the other hand, our results for adenocarcinomas (92.5% of intense staining) diverge from those reported by Santin et al.24 Epidemiological studies have shown that cervical adenocarcinomas are more aggressive than SCC and that HPV18 frequency is higher in adenocarcinomas.25, 38, 39 Interestingly, in a previous study, we have shown that HPV18-immortalized keratinocytes exhibit higher KLK7 transcript levels when compared with normal and HPV16-immortalized ones.9 A caveat in this study was the impossibility to perform the HPV typing in the paraffin-embedded tissues most possibly because of intense DNA degradation, precluding any conclusions regarding the association between hK7 expression and HPV type. A study including a larger number of HPV-typed L-SIL, HSIL, SCC and adenocarcinomas samples is in progress to investigate this important issue.

hK7 is maximally expressed in the stratum granulosum of the skin epithelium and is transported to the differentiated layers by lamellar granules. In this context, hK7 exhibits a predominantly cytoplasmic and membranous staining area displaying some characteristics such as membranous, droplet-like, supranuclear, subnuclear and luminal staining pattern.40–44 Moreover, similarly to ovarian cancer cells, both cervical adenocarcinomas and cervical squamous cell carcinomas have shown cytoplasmic and membranous hK7 staining pattern.23, 24 Consistent with this view, in our study, we observed a similar pattern in SCC and adenocarcinomas as well as in the majority of H-SIL (Figs. 2b2d). Interestingly, most of the hK7 positive cells of cervicitis and L-SIL samples displayed nuclear, cytoplasmic and intercellular space or junction staining. This is the first study reporting the presence of hK7 in the nuclei of cervicitis and L-SIL samples cells. Ongoing studies will determine whether the nuclear and/or cytoplasmic hK7 expression could be associated with HPV type or integration.

Considerable efforts have been spent on identifying novel markers that can be used to accurately predict cervical cancer outcome, mainly adenocarcinomas. Our data suggest the use of hK7 as an additional marker for screening cervical cancer precursor lesions. Further studies including a larger number of samples from different disease stages will be necessary to validate our data. SCC can be prevented by routine cytological screening. However, there is consensus that this method based on Pap testing has important limitations such as the relative high numbers of false-negative results and reduced efficacy in detecting adenocarcinomas.8, 45 For these reasons, the identification of biomarkers that could predict the development of cervical dysplasia and SCC would offer a significant benefit to patients at risk of disease progression. When proven, such biomarker could in addition be used as a tool in the screening of adenocarcinomas treatment and follow-up procedures as well.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors are grateful to Dr. Eleftherios Diamandis (Mount Sinai Hospital, Toronto) for providing the polyclonal antibody for human kallikrein 7 (hK7) and Carlos Ferreira do Nascimento and Severino Ferreira from the Hospital A.C. Camargo for technical assistance. They thank Dr. Enrique Boccardo and Dr. Ana Paula Lepique for critical review of the manuscript. Dr. Lara Termini is a Postdoctoral fellow from Fundação de Amparo à Pesquisa do Estado de São Paulo.

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  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_25046_sm_SuppFig1.tif407KSupporting Information Figure 1. Distribution of hK7 staining according to diagnostic classification. Samples were grouped depending on the staining intensity as negative or weak ([lt] 10% positivity), moderate (10-50% positivity) and intense ([mt]50% positivity). Cerv: Cervicitis; L-SIL: low-grade squamous intraepithelial lesions; H-SIL: high-grade squamous intraepithelial lesions; SCC: squamous cervical carcinomas; ADENO: cervical adenocarcinomas. Numbers on top of each bar correspond to the number of samples in each category.
IJC_25046_sm_SuppFig2.tif392KSupporting Information Figure 2. Distribution of hK7 staining according to diagnostic classification: combining cervicitis and L-SIL in one category. Samples were grouped depending on the staining intensity as negative or weak ([lt] 10% positivity), moderate (10-50% positivity) and intense ([mt]50% positivity). Cerv+L-SIL: cervicitis and low-grade squamous intraepithelial lesions; H-SIL: high-grade squamous intraepithelial lesions; SCC: squamous cervical carcinomas; ADENO: cervical adenocarcinomas. Numbers on top of each bar correspond to the number of samples in each category.

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