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. 2015 Feb 9;96(2):81–86. doi: 10.1111/iep.12115

CYP3A isoforms in Ewing's sarcoma tumours: an immunohistochemical study with clinical correlation

Hamid Zia *, Graeme I Murray †, Carrie A Vyhlidal ‡, J Steven Leeder ‡, Ahmed E Anwar §, Marilyn M Bui , Atif A Ahmed **,✉
PMCID: PMC4459799  PMID: 25670065

Abstract

Ewing's sarcoma is an aggressive malignancy of bone and soft tissue with high incidence of metastasis and resistance to chemotherapy. Cytochrome P450 (CYP) monooxygenases are a family of enzymes that are involved in the metabolism of exogenous and endogenous compounds, including anti-cancer drugs, and have been implicated in the aggressive behaviour of various malignancies. Tumour samples and clinical information including age, sex, tumour site, tumour size, clinical stage and survival were collected from 36 adult and paediatric patients with Ewing's sarcoma family tumours. Tissue microarrays slides were processed for immunohistochemical labelling for CYP3A4, CYP3A5 and CYP3A7 using liver sections as positive control. The intensity of staining was scored as negative, low or high expression and was analysed statistically for any association with patients' clinical information. Four cases were later excluded due to inadequate viable tissue. CYP3A4 staining was present in 26 (81%) cases with high expression noted in 13 (40%) of 32 cases. High expression was significantly associated with distant metastases (P < 0.05). CYP3A5 and CYP3A7 were expressed in 5 and 13 cases respectively (15.6%, 40.6%). There was no association between the expression of CYP3A isoforms and age, sex, tumour size, or location (pelvic or extra-pelvic). None of the biomarkers showed any correlation with overall or disease-free survival. In conclusion, expression of CYP3A isoforms is noted in Ewing's sarcoma tumours and high CYP3A4 expression may be associated with metastasis. Additional studies are needed to further investigate the role of CYP3A4 in the prognosis of these tumours.

Keywords: CYP3A4, cytochrome p450, Ewing's sarcoma, Immunohistochemistry

Ewing's sarcoma family tumours (ESFT) are a group of aggressive malignancies of bone and soft tissue that include Ewing's sarcomas, Askin tumours and primitive neuroectodermal tumours. ESFT are the second most common primary malignant bone tumour after osteosarcoma, accounting for three per cent of all childhood malignancies (Kissane et al. 1983; Grier 1997). The incidence peaks in the second decade, and it is more common in males and Caucasians (Carvajal & Meyers 2005). Histologically they appear as small round cells that exhibit variable neuroepithelial differentiation (Pinto et al. 2011; Choi et al. 2014). These tumours are genetically characterized by gene fusions involving most commonly the EWS gene to the ETS family genes, primarily FLI-1 (Arvand & Denny 2001; Carvajal & Meyers 2005). ESFT are clinically very aggressive in that they have high metastatic tendency and can quickly become resistant to conventional therapy (Grier 1997; Pinkerton et al. 2001). Multimodality treatment has resulted in remarkable improvement in survival of patients with localized disease. However, metastatic tumours are typically refractory to conventional chemotherapy and irradiation, and the presence of metastatic disease at diagnosis is the most important prognostic factor affecting outcome (Grier 1997; Pinkerton et al. 2001; Pinto et al. 2011). One possible reason for the rapid development of resistance to treatment may lie in the expression of proteins or enzymes involved in drug efflux or deactivation of chemotherapeutic agents (Ahmed et al. 2014).

The cytochrome P450 (CYP) monooxygenases are a multigene family of heme-containing, constitutive and inducible enzymes that are involved in phase I metabolic reactions of a wide range of endogenous and xenobiotic compounds, including anti-cancer drugs (Raleigh et al. 1999; van Schaik 2005; Ortiz de Montellano 2013). The CYP isoforms can also activate signalling protein kinases and induce cellular proliferation. They also can oxidize procarcinogens to reactive metabolic intermediates capable of inducing genetic mutations and thus can serve as tumour promoters. Thus, expression of CYP isoforms by cancer cells may influence tumour development, progression and resistance to therapy (Murray 2000; Zordoky & El-Kadi 2009; Panigrahy et al. 2010). Various studies have documented CYP expression in tumour cells opening new avenues into cancer therapeutics (Patterson et al. 1999; Patterson & Murray 2002; McFadyen et al. 2004; Rodriguez-Antona & Ingelman-Sundberg 2006; Panigrahy et al. 2010).

The CYP 3A family is one of the main cytochrome P 450 families and consists of four closely related isoforms: CYP3A4, CYP 3A5, CYP 3A7 and CYP3A43 (de Wildt et al. 1999; Burk & Wojnowski 2004). CYP 3A4 is the major isoform responsible for most CYP3A-mediated actions and is constitutively expressed in the liver and intestine. CYP 3A7 is expressed primarily in foetal livers and subset of adult livers (Leeder et al. 2005; Sim et al. 2005). CYP3A5 is polymorphically expressed in both foetal and adult tissues, predominantly as a result of the presence or absence of the CYP3A5*3 allele, a single nucleotide polymorphism in intron three which produces a truncated protein as a consequence of alternative splicing (Hakkola et al. 2001; Kuehl et al. 2001). CYP3A isoforms expression in neoplastic tissues can maintain tumour growth by metabolic activation of carcinogens and/or procarcinogens, and by activation of protein signalling kinases (Dhaini et al. 2003). They can also cause metabolic deactivation of cancer therapeutic drugs (McFadyen et al. 2004). Expression of CYP3A isoforms was identified in several cancers but has not been previously studied in ESFT. In this study, we looked at the expression profile of CYP3A4, CYP3A5 and CYP3A7 isoforms in ESFT in relation to their biological and clinical behaviour.

Materials and methods

Patients

This study was carried out in accordance with the research protocol approved by the Institutional Review Boards at the Moffitt Cancer Center and the University of South Florida. Patients' tumour samples were collected from the archives of the Pathology Department over a period of 13 years (1995–2007). All cases with diagnoses of Ewing's sarcoma, primitive neuroectodermal tumour and Askin tumour were included in the study. The diagnoses were confirmed by a designated sarcoma pathologist for this study including re-examination of the H&E slides and ancillary studies. Confirmatory ancillary studies performed included combination of diffuse or focal cytoplasmic PAS positivity and strong diffuse membranous positivity for CD99. Translocation analysis was performed when necessary, for example in tumours of atypical location or atypical morphology. Pertinent clinical data compiled include patient's age, sex, tumour site, tumour size, clinical stage and survival (overall and disease-free survival) (Table1). All tissues were obtained as either early diagnostic specimen or during resection that was performed after initiation chemotherapy. Patients were clinically followed up to 14 years. At the end of the follow-up period, patients were grouped into two categories, as alive or dead. Survival periods were calculated in days, categorized as overall survival (OS) and disease-free survival (DFS).

Table 1.

Clinicopathological information of patients and data stratification, N = 36

Clinicopathologic parameters Data stratification N (%)
Age (years) ≤20 10 (27.8)
>20 26 (72.2)
Sex Males 23 (63.9)
Females 13 (36.1)
Patients' outcome Alive 19 (52.8)
Dead 17 (47.2)
Tumour site Primary skeletal 20 (55.5)
Primary extraskeletal 10 (27.8)
Metastatic 6 (16.7)
Tumour size <10 cm 16 (44.4)
≥10 cm 6 (16.7)
Unknown 14 (38.9)
Clinical stage Localized 12 (33.3)
Regional extension 9 (25.0)
Distant metastasis 9 (25.0)
Unknown 6 (16.7)
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Tissue microarray preparation

After confirming the diagnosis, the most representative sections and the corresponding portions (0.6 mm diameter) of the paraffin-embedded blocks were selected for the construction of tissue microarray blocks according to a previously described method (Schlauder et al. 2008). Inclusion criteria included adequate tumour volume, adequate tissue blocks and clinical data. Two samples were obtained from two representative areas of a given tumour block and placed adjacent to each other in tissue microarray (TMA). Because of extensive sectioning, adequate viable stainable and interpretable tissue was becoming scant and was lost in a few cases.

Immunohistochemistry

Sections were stained for CYP3A4 and CYP3A5 with monoclonal antibodies which have been produced in the Murray2 laboratory. CYP3A4 monoclonal antibody (Murray et al. 1987, 1988) was produced with partially purified CYP3A4 peptide as the immunogen while CYP3A5 monoclonal antibody (Kumarakulasingham et al. 2005) was developed using a C-terminal peptide as the immunogen. Immunohistochemistry for each antibody was performed with the biotin-free Dako Envision™ system (Dako, Ely, UK) with a Dako autostainer. Sections of the tissue microarray were dewaxed in xylene and rehydrated in alcohol. An antigen retrieval step was performed which consisted of microwaving the sections fully immersed in 10 mM citrate buffer at pH6.0 for 20 min in an 800W microwave oven operated at full power. The sections were then allowed to cool to room temperature prior to application of the primary antibody. Each primary antibody was applied as undiluted tissue culture supernatant. Omitting the primary monoclonal antibody from the immunohistochemical procedure and replacing it with antibody diluent acted as a negative control.

CYP3A7 (F19 P2 H2) antibody was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Automated immunohistochemistry was performed with a Leica Bond Max Instrument (Leica, Richmond, IL, USA) utilizing a Bond Polymer Refine Detection system that gives a brown colour staining (Joan Whiting, MT). After heat antigen retrieval methods, deparaffinized slides were sequentially incubated with the primary antibody, a secondary antibody, a polymer conjugate and a colouring reagent. Normal liver sections known to express CYP3A4, CYP3A5 and CYP3A7 were used as a positive control. Negative controls were treated similarly except for omission of the primary antibodies.

Analysis of results

Stained slides were analysed by low (×100) and high magnifications (×400) and compared to normal positive controls. The extent of immunohistochemical (IHC) expression was scored by two pathologists using the following system: 0 (negative) = no staining; 1+ (low level of expression) = weak staining that is less than that of positive controls and/or staining that is only clearly visible by high magnification; and 2+ (high level of expression) = strong staining that is equal to or more than that of positive controls and/or staining that is clearly visible by low magnification. The staining scores of the two sections of each patient's tumour were averaged into one score.

Kaplan–Meier survival curves were also plotted to demonstrate difference in survival between high and low/negative IHC expression groups of all the selected proteins. The effects of clinicopathologic parameters and IHC expression of the studied proteins on overall and disease-free survival were examined by univariate cox proportional hazards model.

Results

Patients

Of the 36 patients, 23 were males and 13 were females (M:F = 1.8). Ages ranged from 12 to 72 years. Twenty six (72.2%) of the patients were older than 20 years, and 10 (27.8%) of the patients were of the paediatric age group (≤20 years). Diagnostic specimens were from primary locations in 30 patients and from diagnostic metastatic site biopsies in six patients. Patients were followed clinically up to 14 years from the primary diagnosis, with a minimal follow-up of 25 days and median of 759 days. At the end of the follow-up period, 17 (47.2%) of the patients were dead and 19 (52.8%) were alive. Overall survival (OS) ranged from 25 to 5065 days and DFS from 25 to 4224 days.

Tumours

Tumours were considered primary (skeletal or extra-skeletal) in 30 patients and only six patients presented with metastatic disease. Primary tumours were located in the bone in 20 (55.5%) of the patients and were extra-skeletal in 10 (27.8%) of the patients. Of the skeletal tumours, four cases were located in the chest wall (Askin tumour). Tumour size was measured in 22 patients and ranged from 1.2 to 34.5 cm. Margin statuses of the resection specimens were known in 21 patients. All except one had negative margins. Clinical stage of the tumours was determined in 30 cases and was grouped into localized (12 cases), regional extension (9 cases) and distant metastasis (9 cases) (Table1). The latter group included six patients who presented with metastatic disease and three patients who developed metastasis later during follow-up.

Immunostaining results

Of the 36 cases of Ewing's sarcoma family of tumours selected, four variable cases were later excluded due to inadequate viable tissue. Positive staining of CYP3A4, CYP3A5 and CYP3A7 was discerned to be cytoplasmic. CYP3A4 staining was identified in 26 (81%) cases, with high expression present in 13 (40%) of 32 cases (Figure1). High expression of CYP3A4 was significantly associated with distant metastases, and negative or low expression was observed in localized and/or regional extension of disease (P < 0.05). There was no association of high CYP3A4 expression with age, sex, tumour size or location (pelvic or extra-pelvic) (table2). We did not find a statistically significant difference in the overall and disease-free survival between the low and high CYP3A4 expression groups (Figure2).

Figure 1.

Figure 1

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Immunohistochemical staining of CYP3A4 (a: ×200), CYP3A5 (b: ×200) and CYP3A7 (c: ×200) in Ewing's sarcoma tumours revealing staining scores of 2, 1 and 2 respectively. Similar staining was noted in the liver tissue that served as positive control for CYP3A4 and CYP3A5 (d and e: ×200 respectively) while the negative control did not display any staining (f: ×200).

Table 2.

Association between CYP3A4 and patients' demographic and clinical data (n = 32)

Levels Negative/Low, n = 15 High, n = 17 P-value
Sex
 Female, n (%) 6 (40.0) 5 (29.41) 0.5291
 Male 9 (60.0) 12 (70.59)
Age/Year
 ≤20 4 (26.67) 5 (29.41) 1.0000
 >20 11 (73.33) 12 (70.59)
Stage
 Distant mets 0 (0.0) 7 (53.85) 0.0070*
 Localized 6 (46.15) 4 (30.77)
 Regional direct extension 7 (53.85) 2 (15.38)
Tumour size
 Mean ± SD 6.6 ± 4.3 6.4 ± 3.9 0.9232
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*

Fisher's exact test significant at p = 0.05. Of the initial cohort of 36 patients, only 32 cases had adequate tissue suitable for CYP3A4 staining and interpretation. These included seven patients with the ‘distant metastasis’ group, five of whom initially presented with metastasis.

Figure 2.

Figure 2

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Kaplan–Meier survival curve displaying the effect of high CYP3A4 expression on disease-free survival.

CYP3A5 and CYP3A7 (Figure1) were expressed in 5 and 13 cases (15.6% and 40.6%) respectively and were not associated with age, sex, tumour size, tumour location, clinical stage, or overall or disease-free survival. CYP3A7 revealed strong staining corresponding to high expression in all the cases while CYP3A5 had only weak expression (Figure1).

Discussion

This study demonstrates the expression of CYP3A isoforms in ESFT thus suggesting that they may have a role in ESFT tumour progression. CYP3A isoforms affect cellular metabolic and signalling changes that ultimately lead to tumour progression and metastasis (Patterson et al. 1999; Raleigh et al. 1999; Panigrahy et al. 2010; Ortiz de Montellano 2013). Expression of CYP3A isoforms in cancer cells may also lead to local deactivation of chemotherapeutic agents resulting in resistance and recurrence of metastatic disease (Murray et al. 1993). Compared to other isoforms, CYP3A4 was more prevalent in ESFT cases in this study and high CYP3A4 expression was associated with metastatic disease. CYP3A4 is involved in metabolism of various anti-cancer drugs that are used for the treatment of ESFT, such as cyclophosphamide, doxorubicin, dactinomycin, vincristine, ifosfamide, topotecan and etoposide (Dhaini et al. 2003; McFadyen et al. 2004; Rodriguez-Antona & Ingelman-Sundberg 2006). Thus, expression of CYP3A4 in ESFT may allow tumour cells to escape the effect of chemotherapy and allows for tumour progression.

Overexpression of CYP3A isoforms was previously demonstrated to be associated with more aggressive behaviour in breast cancer, osteosarcoma and a variety of other benign and malignant lesions (Hughes et al. 1999; Murray et al. 1999; Dhaini et al. 2003; Tayeb et al. 2003; Kumarakulasingham et al. 2005). High expression of CYP3A4 in osteosarcomas was a statistically significant marker of metastasis and overall poor prognosis (Dhaini et al. 2003). Similarly, in breast cancer, decreased response to docetaxel and lymph node metastasis was observed in patients with breast cancer whose tumours possessed high CYP450 mRNA expression levels (Murray et al. 2010). The expression of CYP3A4/5 was associated with poor tumour differentiation and occurrence of lymph node metastasis, with strong expression of CYP3A4/5 in advanced T-stages (Haas et al. 2006). Thus, it is of no surprise that CYP3A4 expression in ESFT was associated with metastasis. The absence of association with patient survival suggests that other factors play a role in determining the outcome. Targeting CYP3A4 may offer hope for cure in patients with metastatic or chemoresistant ESFT tumours (Scotlandi et al. 2009).

In conclusion, CYP3A isoforms are expressed in a subset of patients with ESFT, and expression of CYP3A4 may be associated with metastatic disease. Although this conclusion is based on immunohistochemical studies on tissue microarray slides, it warrants due thoughtfulness and consideration for the role of these important cellular metabolic enzymes in ESFT. The study is limited by small patient population size and the small size of sampled tumour tissue available for staining in the microarray slide. Different results may be obtained if whole-slide tumour cases from larger number of patients are studied. Because of the role of these biomarkers in cancer progression in general, additional studies are needed to confirm our findings and clarify the role of cytochrome P450 enzymes in ESFT.

Acknowledgments

Hamid Zia: Performed research analysis and drafted initial manuscript Graeme Murray: Performed essential experiments Carrie Vyhlidal: Supplied essential reagents Steven Leeder: Supplied essential reagents Ahmed Anwar: Performed biostatistical analysis Marilyn Bui: Supplied patients data and materials Atif Ahmed: Designed research concept, analyzed data and finalized the manuscript.

Conflict of interest

Authors do not have any conflicts of interest to report.

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