Oral Diseases (2013) doi:10.1111/odi.12216 © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd All rights reserved www.wiley.com
ORIGINAL ARTICLE
Salivary and serum level of CYFRA 21-1 in oral precancer and oral squamous cell carcinoma K Rajkumar1, R Ramya1, G Nandhini1, P Rajashree1, A Ramesh Kumar1, S Nirmala Anandan2 1
Department of Oral and Maxillofacial pathology, SRM Dental College, SRM University, Chennai; 2Department of Biochemistry, SRM Dental College, SRM University, Chennai, India
BACKGROUND: CYFRA 21-1, a constituent of the intermediate filament proteins of epithelial cells, is known to be increased in many cancers. This study was designed to estimate the levels of salivary and serum CYFRA 21-1 in patients with oral precancer and oral squamous cell carcinoma (OSCC) and compare them with healthy controls. MATERIALS AND METHODS: Each group comprised of 100 subjects. Saliva and blood samples were collected from patients with OSCC, premalignant subjects, and normal healthy subjects. Serum and salivary CYFRA 21-1 levels were measured by enzyme-linked immunosorbent assay. Appropriate statistical tests were employed to assess diagnostic potency of CYFRA 21-1. RESULTS: We found a significant increase in CYFRA 21-1 level in OSCC compared with PML and healthy subjects. Salivary CYFRA 21-1 levels in OSCC was threefold higher when compared to serum levels. PML group showed increased salivary CYFRA 21-1 when compared to control subjects, but it was significantly lower compared with OSCC. Receiver operator characteristic curve analysis showed salivary CYFRA 21-1 to have superior sensitivity in detecting OSCC compared with serum CYFRA 21-1. CONCLUSIONS: The outcome of this study suggests that salivary CYFRA 21-1 can be utilized as a biomarker in early detection of oral cancer. Oral Diseases (2013) doi: 10.1111/odi.12216 Keywords: oral cancer; diagnosis; CYFRA 21-1; premalignant lesion; squamous cell carcinoma
Correspondence: Dr. Krishnan Rajkumar, Department of Oral and Maxillofacial pathology, SRM Dental College, SRM University, Ramapuram campus, Chennai 600089, India. Tel: +91 044 22490526, Fax: +91 044 22492429, E-mail: [email protected] Received 7 August 2013; revised 29 October 2013; accepted 10 November 2013
Introduction Oral cancer is one of the most aggressive malignancies worldwide. It is ranked as the sixth leading cause of cancer mortality worldwide and the second leading cause of cancer mortality in India (Petersen, 2009; Warnakulasuriya, 2009). Due to its high propensity for local invasion, distant metastases, and a lack of early detection methods when diagnosed, more than two-thirds of cases demonstrate advanced or unresectable forms of the disease. Thus, in spite of advancement in treatment strategies, the 5-year survival rate for oral cancer patients remains only 50% for the past three decades (Gomez et al, 2010). The pathogenesis of oral cancer is a complex, multistage, and multigenetic process, of which the precise mechanisms are not yet fully understood. Most of the oral cancers are oral squamous cell carcinoma (OSCC). Of note, it is reported that there is a transition from premalignant lesion (PML) to malignancy for over a period of time (Shiu and Chen, 2003; Liu et al, 2010). This makes it necessary to constantly screen the population where there is high incidence of these lesions due to tobacco chewing/ smoking habits. Surveys in India have shown that about 80% of oral carcinomas are preceded by oral potentially malignant disorders, most often persistent leukoplakia (most common premalignant lesion) and oral submucous fibrosis (OSMF), which predominantly affects the people of South-East Asian origin (Gupta et al, 1989; Cheng et al, 2004; Lalli et al, 2008). Hence, it would be of great aid if effective diagnostic/screening marker that monitors premalignant lesion were available for routine use. Many studies have focused on discovering a biomarker for screening OSCC in saliva, so as to develop a noninvasive and simple method of screening. Although several markers have been discovered in saliva in the recent years (Liu and Duan, 2012), their validity for routine use as a marker has not been much focused. Among the various salivary biomarkers of OSCC, cytokeratin fraction 21-1 (CYFRA 21-1) has been reported to be potent marker (Nagler et al, 2006). Cyfra 21-1 is a well-accepted tumor marker with high sensitivity and specificity in nonsmall cell lung cancer, especially squamous cell carcinoma (independent prognostic factor) (Nisman et al, 1998;
Salivary CYFRA 21-1- a marker for oral cancer
K Rajkumar et al
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Kawaguchi et al, 2000). While there are reports that CYFRA 21-1 is a useful oral tumor marker (Nagler, 2009), to our knowledge, the clinical utility of this marker in saliva as a screening tool in potentially malignant condition to detect OSCC has not been addressed. In this study, we have validated the diagnostic utility of CYFRA 21-1 in differential diagnosis of premalignant (leukoplakia & OSMF) and malignant oral condition (OSCC) in serum and saliva. Furthermore, the study also compared the levels of CYFRA 21-1 with histological gradation and cancer staging in patients with OSCC.
Materials and methods This study was approved by the Scientific Advisory Committee and Institutional Ethical Committee of SRM University, Chennai. According to ethical principles, including the World Medical Association Declaration of Helsinki (version, 2002), a written and informed consent was obtained from all the study participants before drawing blood and collecting saliva. Study subjects recruitment The recruitment of study subjects was carried out at outpatient department of SRM dental college, referrals from SRM General Hospital and private dental clinics, Chennai during January 2009 and December 2012. The demographic details and information on previous history were collected. Subjects with infectious diseases during 1 month before saliva sampling, active dental abscesses, systemic illness, and collagen vascular diseases were excluded from the study. None of the lesions had been treated in any manner prior to sample collection. None of the control participants had oral lesions. Pregnant and lactating subjects were also excluded. After registering the eligible patients, the histopathological examination was carried out for premalignant and OSCC subjects. A standard tissue biopsy was taken from each patient. The diagnosis was confirmed by two pathologists independently. The biopsies were further classified based on histopathological gradation. Sample collection Saliva samples were collected between 9 and 11 A.M under non-stimulatory condition. Participants were asked to refrain from eating, chewing, and drinking at least one hour before collection. 3–5 ml of salivary samples from patients with oral carcinoma and premalignant lesion was collected before any therapeutic procedure. Following collection, the saliva was immediately centrifuged to remove cell debris. Supernatants were then stored at 80°C. 2 ml of peripheral blood was drawn from all study subjects through venipuncture. Blood was transferred to an empty vacutainer. Serum was then collected by centrifuging the coagulated blood and stored at 80°C until further use. Estimation of CYFRA 21-1 Concentrations of salivary and serum CYFRA 21-1 were quantified by commercially available ELISA Kit (Syntron Bioresearch In., Carlsbad, CA, USA). The assay was
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carried out according to the manufacturer’s instruction. Absorbency was 620 nm and measured by microplate reader (RoboniK ELISA plate reader). The detection range of the kit was from a minimum of 0.5 ng to a maximum of 50 ng ml 1. The results were expressed as ng ml 1 of saliva or serum. Statistical analysis The data were analyzed using SPSS software, version 21.0 (SPSS, Chicago, IL, USA). Nonparametric Mann– Whitney U-tests were performed to find the significance of the observed differences in OSCC and PML groups. A P value of 10.4, salivary CYFRA 21-1 shows 75% specificity and sensitivity in differentiating PML from OSCC. On the other hand, a cutoff value of 2.5 ng ml 1 in serum CYFRA 21-1 level showed 90% specificity, while the sensitivity was reduced to 60%. Nevertheless, with salivary CYFRA 21-1, we evaluated cutoff value with 95% specificity in healthy control and found it to be >7.91. With this cutoff, the sensitivity was found to be
Figure 2 The levels of CYFRA 21-1 compared between serum and saliva of healthy control, PML and OSCC subjects. The results are expressed as mean SEM. The statistical significance is shown as * when compared within group, # when compared with healthy control group, and $ when compared with PML group. P < 0.05 was considered to be statistically significant
83.6% in detecting the OSCC group. Curve comparing the diagnostic ability of salivary and serum CYFRA 21-1 in detecting OSCC using ROC curve is illustrated (Figure 3). A significant difference (P < 0.05) was noted between the Oral Diseases
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Table 2 Level of serum and salivary CYFRA 21.1 levels in different study groups
Study subjects Healthy Control (n = 100) Premalignant lesion subjects (n = 100) Oral squamous cell carcinoma (n = 100)
Serum CYFRA 21.1 (ng ml 1)
Salivary CYFRA 21.1 (ng ml 1)
1.07 0.073 1.50 0.08a
3.06 0.25c 8.15 0.50ac
5.14 0.38ab
17.46 1.46abc
The levels of serum and salivary CYFRA 21.1 were assessed by ELISA in all the study subjects. The concentrations are expressed in nanogram per milliliter, and the values represent mean SEM. a P < 0.05 when compared to healthy control group, bwhen compared to premalignant lesion group, and cwhen compared between serum saliva.
AUC for salivary (0.899) and serum CYFRA 21-1 (0.865) based on pairwise comparison of ROC curves. ROC analysis demonstrates that salivary CYFRA- 21-1 was superior to serum CYFRA 21-1, specifically in differentially diagnosing OSCC with that of PML. CYFRA 21-1 levels based on histological grading and clinical staging For 100 patients with OSCC, CYFRA 21-1 concentrations were compared according to histological differentiation (Table 1, Figure 4a,b). The median and interquartile range of serum CYFRA 21-1 concentration were 4.08 (0.9– 20.4), 3.3 (0.41–19.3) and 5.6 (0.32–14.5) ng ml 1 for well-differentiated (WD), moderately differentiated (MD), and poorly differentiated (PD) lesions, respectively (Figure 4a). The serum CYFRA 21-1 concentration did not show any significant difference between the histological grading in OSCC. Median and interquartile range of salivary CYFRA 21-1 concentrations were 11.8 (6.8–28.4) for
WD, 13.9(8.2–89.2) for MD, and 18.3 (9.7–110.3) for PD lesion, respectively (Figure 4b). An overall tendency toward significant (P < 0.001) increase in salivary CYFRA 21-1 concentration was observed for PD lesion. No significant difference was evident for salivary CYFRA 21-1 concentration between WD and MD lesion. Also, no significant difference with salivary CYFRA 21-1 was found depending on the histological grading in PML (data not shown). The level of CYFRA 21-1 in serum and saliva was also compared based on the clinical staging of OSCC patients (Figure 4c,d). In serum, the median and interquartile range for stage I is 2.60 (0.32–6.40), stage II is 3.56 (0.41– 6.19), stage III is 6.97 (3.97–19.3), and stage IV is 9.29 (4.19–20.49). Similarly for saliva, the median and interquartile range for stage I is 15.35 (3.56–24.95), stage II is 14.5 (0.31–29.45), stage III is 16.4 (9.2–30.34), and stage IV is 29.5 (18.35–110.3), respectively. Interestingly, serum CYFRA 21-1 levels were significantly increased in stage III and IV (P < 0.01). No difference was observed between stage I and stage II and between stage III and IV. On the other hand, in saliva, significant increase was found only with stage IV (P < 0.001).
Discussion This is the first study comparing the use of CYFRA 21-1 as a biomarker in saliva for differential diagnosis of premalignant and malignant oral carcinoma in a population with high incidence of oral cancer. Although both serum and salivary CYFRA 21-1 levels were significantly higher for OSCC compared with PML, salivary CYFRA 21-1 showed threefold increase compared with serum CYFRA 21-1. Furthermore, the validation of CYFRA 21-1 levels
Figure 3 ROC curve analysis of CYFRA 21-1 marker in saliva and serum for predicting OSCC when compared to PML Oral Diseases
Salivary CYFRA 21-1- a marker for oral cancer
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(a)
(c)
25
* (P * < 0.001) (P < 0.001)
15
ng ml–1
ng ml–1
20
10 5 0
(b)
(P < 0.001)
(P < 0.01)
Stage I
Stage II
Stage III
Stage IV
(d)
100
(P * < 0.001)
ng ml–1
ng ml–1
80 60 40 20
0
Stage I
Stage II
Stage III
Stage IV
Figure 4 The levels of CYFRA 21-1 compared in serum and saliva of OSCC subjects based on histological grading (a and b) [well differentiated (WD), moderately differentiated (MD), and poorly differentiated (PD)] and based on clinical staging (c and d). Each dot represents CYFRA 21-1 level of each individual subject, and the horizontal line represents mean value. The statistical significance is shown as * when compared to healthy control and # when compared to PML subjects. P < 0.05 was considered to be statistically significant
with ROC curve revealed salivary CYFRA 21-1 to have high sensitivity for OSCC. CYFRA 21-1 is a well-established monoclonal antibody that reacts exclusively with cytokeratin-19. Cytokeratin family consists of 19 different polypeptides, which have been numbered 1–19 (Moll et al, 1982). These cytokeratins are not randomly distributed in epithelia, but appear to be characteristic for certain types of epithelial differentiation (Gasparoni et al, 2005). Cytokeratin-19 is an acidic (type I) subunit expressed in all simple epithelia and in carcinomas which arise from them (Broers et al, 1985, 1988), Hence, it was found to be a potential marker in many squamous cell carcinomas. CYFRA 21-1 has been reported to be an established marker for lung cancer (Nisman et al, 1998). In addition, it is also shown to be a potent marker for head and neck cancer, gastric cancer, uterine cancer, and esophageal cancer (Doweck et al, 1995; Nakata et al, 1996; Bonfrer et al, 1997; Yamamoto et al, 1997). The utility of CYFRA 21-1 for OSCC was first demonstrated by Kurokawa et al (1997). They showed significant increase in serum CYFRA 21-1 of patients with OSCC compared with control and benign oral tumor subjects. Later many studies reported high levels of serum CYFRA 21-1 in OSCC with varying sensitiv-
ity and specificity. Some studies claimed serum CYFRA 21-1 to be poor diagnostic marker in detecting OSCC because of low sensitivity and specificity (Hamakawa et al, 1998; Yen et al, 1998; Feng et al, 2010), while other studies showed it to be accurate diagnostic and prognostic marker for OSCC (Nagler et al, 2006; Zhong et al, 2007b). This discrepancy may be due to the difference in the methodology employed in measuring CYFRA 21-1 and study design. In accordance with Nagler et al (2006) and Zhong et al (2007a), we also found higher levels of serum CYFRA 21-1 in OSCC subjects compared with controls. According to Sugama et al (1994), increased serum CYFRA 21-1 is the net result of cytokeratin release due to cell lysis or necrosis. Moreover, during epithelial transition to malignancy, there is an increase in the degradation of cytokeratin which in turn results in release of large amount of cytokeratin fragments in the local environment (Ausch et al, 2010). Taking this into account, it is more likely for saliva, the local environment of OSCC to show high levels of CYFRA 21-1. In concordance with this hypothesis, Nagler et al (2006) found fourfold increase in amount of CYFRA 21-1 in saliva compared with serum illustrating saliva to be best medium for OSCC diagnosis Oral Diseases
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(Nagler et al, 2006). This was again proved with another study where salivary CYFRA 21-1 levels were significantly higher in OSCC subjects (Zhong et al, 2007a). Concordant to the above reports, our study showed threefold higher level of CYFRA 21-1 in saliva when compared to serum. From the present study, we also find significantly higher CYFRA 21-1 levels in saliva of PML and not in serum compared with control. This shows that there occurs some change in these oral lesions, which is immediately reflected in the saliva rather in serum. The ROC curve analyses further confirmed salivary CYFRA 21-1 to possess higher sensitivity and specificity than serum CYFRA 21-1. Although the specificity of serum CYFRA 21-1 was more than 85%, their sensitivity was low to be considered for diagnostic utility. Moreover, high sensitivity is the hallmark for a marker to be used for screening test, from the present study it is evident that salivary CYFRA 21-1 is more useful as a screening marker rather than serum CYFRA 21-1 levels. Also, we found salivary level to be significantly higher with poorly differentiated lesions of OSCC, but the same was not reflected in serum levels. However, when compared based on clinical staging of cancer, we found CYFRA 21-1 to be increased in stage IV in serum and saliva. This shows that CYFRA 21-1 levels are more increased toward the end stage of cancer, and therefore, CYFRA 21-1 level is shown to increase with the severity of disease as reported by others (Brockmann et al, 2000). Of late, reports show that there is decline in serum CYFRA 21-1 after chemoradiotherapy in esophageal squamous cell carcinoma, suggesting it as a prognostic marker (Yi et al, 2009; Yan et al, 2012). Although it is well established that radiation affects salivary flow rate and secretion, it would be of interest to know the status of salivary CYFRA 21-1 in all types of OSCC, so that it can be considered for prognostic marker as well. The main limitation of this study is that it did not include other PML lesion such as erythroplakia and oral lichen planus in PML group. The reason for their exclusion is their rare occurrence, and moreover, leukoplakia and OSMF are the one which are stated to transit to malignant form at a high rate compared with others. So, this should not undervalue the outcome of the study. It is also important to note the effect that tobacco smoking/ chewing has on CYFRA 21-1 levels. But it could not account for the difference in CYFRA 21-1 levels between the patients with cancer and the PML subjects, as the numbers of smokers among these two groups were similar, and moreover, it is reported that smoking has no effect on CYFRA 21-1 concentration (Kao et al, 1999). Therefore, in healthy smokers also, it should not affect the salivary or serum CYFRA 21-1 levels. In summary, the significantly higher levels of salivary CYFRA 21-1 in OSCC is encouraging in light of the many advantages of saliva measurement in comparison with serum analysis. This suggests that salivary CYFRA 21-1 may be considered for diagnostic tool as it is often very difficult to distinguish clinically between a premalignant and OSCC by visual screening alone. Accordingly, such an analysis may turn into a valuable screening tool as it might save many unnecessary biopsies and will also
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allow easy adherence of patients for constant follow-up for screening in regions with high incidence of PML due to tobacco intake. Acknowledgements The authors thank the patients and healthy volunteers in the study for their willingness to contribute to medical research. The statistical suggestion by Dr.C. Ponnuraja (Biostatistics Department, National institute of Research in Tuberculosis) is acknowledged.
Author contributions K. Rajkumar conceived and designed the study, interpreted the data and revised the manuscript. R. Ramya was involved in design, sample collection and data interpretation, G. Nandhini did sample collection, carried out ELISA, data management, P. Rajashree did data analysis, statistics, interpretation and manuscript writing, K. A. Ramesh contributed to sample collection and A. S. Nirmala was involved in interpretation and revision of manuscript.
Conflict of interest The authors declare no conflict of interests.
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Gupta PC, Bhonsle RB, Murti PR, Daftary DK, Mehta FS, Pindborg JJ (1989). An epidemiologic assessment of cancer risk in oral precancerous lesions in India with special reference to nodular leukoplakia. Cancer 63: 2247–2252. Hamakawa H, Bao Y, Takarada M, Fukuzumi M, Tanioka H (1998). Cytokeratin expression in squamous cell carcinoma of the lung and oral cavity: an immunohistochemical study with possible clinical relevance. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85: 438–443. Kao CH, Hsieh JF, Ho YJ, Tsai SC, Lee JK (1999). Cytokeratin fragment 19 (CYFRA 21-1) in healthy smokers. Anticancer Res 19: 4545–4546. Kawaguchi H, Ohno S, Miyazaki M et al (2000). CYFRA 21-1 determination in patients with esophageal squamous cell carcinoma: clinical utility for detection of recurrences. Cancer 89: 1413–1417. Kurokawa H, Yamashita Y, Tokudome S, Kajiyama M (1997). Combination assay for tumor markers in oral squamous cell carcinoma. J Oral Maxillofac Surg 55: 964–966. Lalli A, Tilakaratne WM, Ariyawardana A et al (2008). An altered keratinocyte phenotype in oral submucous fibrosis: correlation of keratin K17 expression with disease severity. J Oral Pathol Med 37: 211–220. Liu J, Duan Y (2012). Saliva: a potential media for disease diagnostics and monitoring. Oral Oncol 48: 569–577. Liu W, Wang YF, Zhou HW, Shi P, Zhou ZT, Tang GY (2010). Malignant transformation of oral leukoplakia: a retrospective cohort study of 218 Chinese patients. BMC Cancer 10: 685. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R (1982). The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31: 11–24. Nagler RM (2009). Saliva as a tool for oral cancer diagnosis and prognosis. Oral Oncol 45: 1006–1010. Nagler R, Bahar G, Shpitzer T, Feinmesser R (2006). Concomitant analysis of salivary tumor markers–a new diagnostic tool for oral cancer. Clin Cancer Res 12: 3979–3984. Nakata B, Chung YS, Kato Y et al (1996). Clinical significance of serum CYFRA 21-1 in gastric cancer. Br J Cancer 73: 1529–1532. Nisman B, Lafair J, Heching N et al (1998). Evaluation of tissue polypeptide specific antigen, CYFRA 21-1, and carcinoembryonic antigen in nonsmall cell lung carcinoma: does the
combined use of cytokeratin markers give any additional information? Cancer 82: 1850–1859. Petersen PE (2009). Global policy for improvement of oral health in the 21st century–implications to oral health research of World Health Assembly 2007, World Health Organization. Commun Dent Oral Epidemiol 37: 1–8. Shiu MN, Chen TH (2003). Intervention efficacy and malignant transformation to oral cancer among patients with leukoplakia (Review). Oncol Rep 10: 1683–1692. Sugama Y, Kitamura S, Kawai T et al (1994). Clinical usefulness of CYFRA assay in diagnosing lung cancer: measurement of serum cytokeratin fragment. Jpn J Cancer Res 85: 1178– 1184. Warnakulasuriya S (2009). Global epidemiology of oral and oropharyngeal cancer. Oral Oncol 45: 309–316. Yamamoto K, Oka M, Hayashi H, Tangoku A, Gondo T, Suzuki T (1997). CYFRA 21-1 is a useful marker for esophageal squamous cell carcinoma. Cancer 79: 1647–1655. Yan HJ, Wang RB, Zhu KL et al (2012). Cytokeratin 19 fragment antigen 21-1 as an independent predictor for definitive chemoradiotherapy sensitivity in esophageal squamous cell carcinoma. Chin Med J (Engl) 125: 1410–1415. Yen TC, Lin WY, Kao CH, Cheng KY, Wang SJ (1998). A study of a new tumour marker, CYFRA 21-1, in squamous cell carcinoma of the head and neck, and comparison with squamous cell carcinoma antigen. Clin Otolaryngol Allied Sci 23: 82–86. Yi Y, Li B, Wang Z, Sun H, Gong H, Zhang Z (2009). CYFRA21-1 and CEA are useful markers for predicting the sensitivity to chemoradiotherapy of esophageal squamous cell carcinoma. Biomarkers 14: 480–485. Zhong LP, Zhang CP, Zheng JW, Li J, Chen WT, Zhang ZY (2007a). Increased Cyfra 21-1 concentration in saliva from primary oral squamous cell carcinoma patients. Arch Oral Biol 52: 1079–1087. Zhong LP, Zhu HG, Zhang CP, Chen WT, Zhang ZY (2007b). Detection of serum Cyfra 21-1 in patients with primary oral squamous cell carcinoma. Int J Oral Maxillofac Surg 36: 230–234.
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Oral Diseases
ORIGINAL ARTICLE
Salivary and serum level of CYFRA 21-1 in oral precancer and oral squamous cell carcinoma K Rajkumar1, R Ramya1, G Nandhini1, P Rajashree1, A Ramesh Kumar1, S Nirmala Anandan2 1
Department of Oral and Maxillofacial pathology, SRM Dental College, SRM University, Chennai; 2Department of Biochemistry, SRM Dental College, SRM University, Chennai, India
BACKGROUND: CYFRA 21-1, a constituent of the intermediate filament proteins of epithelial cells, is known to be increased in many cancers. This study was designed to estimate the levels of salivary and serum CYFRA 21-1 in patients with oral precancer and oral squamous cell carcinoma (OSCC) and compare them with healthy controls. MATERIALS AND METHODS: Each group comprised of 100 subjects. Saliva and blood samples were collected from patients with OSCC, premalignant subjects, and normal healthy subjects. Serum and salivary CYFRA 21-1 levels were measured by enzyme-linked immunosorbent assay. Appropriate statistical tests were employed to assess diagnostic potency of CYFRA 21-1. RESULTS: We found a significant increase in CYFRA 21-1 level in OSCC compared with PML and healthy subjects. Salivary CYFRA 21-1 levels in OSCC was threefold higher when compared to serum levels. PML group showed increased salivary CYFRA 21-1 when compared to control subjects, but it was significantly lower compared with OSCC. Receiver operator characteristic curve analysis showed salivary CYFRA 21-1 to have superior sensitivity in detecting OSCC compared with serum CYFRA 21-1. CONCLUSIONS: The outcome of this study suggests that salivary CYFRA 21-1 can be utilized as a biomarker in early detection of oral cancer. Oral Diseases (2013) doi: 10.1111/odi.12216 Keywords: oral cancer; diagnosis; CYFRA 21-1; premalignant lesion; squamous cell carcinoma
Correspondence: Dr. Krishnan Rajkumar, Department of Oral and Maxillofacial pathology, SRM Dental College, SRM University, Ramapuram campus, Chennai 600089, India. Tel: +91 044 22490526, Fax: +91 044 22492429, E-mail: [email protected] Received 7 August 2013; revised 29 October 2013; accepted 10 November 2013
Introduction Oral cancer is one of the most aggressive malignancies worldwide. It is ranked as the sixth leading cause of cancer mortality worldwide and the second leading cause of cancer mortality in India (Petersen, 2009; Warnakulasuriya, 2009). Due to its high propensity for local invasion, distant metastases, and a lack of early detection methods when diagnosed, more than two-thirds of cases demonstrate advanced or unresectable forms of the disease. Thus, in spite of advancement in treatment strategies, the 5-year survival rate for oral cancer patients remains only 50% for the past three decades (Gomez et al, 2010). The pathogenesis of oral cancer is a complex, multistage, and multigenetic process, of which the precise mechanisms are not yet fully understood. Most of the oral cancers are oral squamous cell carcinoma (OSCC). Of note, it is reported that there is a transition from premalignant lesion (PML) to malignancy for over a period of time (Shiu and Chen, 2003; Liu et al, 2010). This makes it necessary to constantly screen the population where there is high incidence of these lesions due to tobacco chewing/ smoking habits. Surveys in India have shown that about 80% of oral carcinomas are preceded by oral potentially malignant disorders, most often persistent leukoplakia (most common premalignant lesion) and oral submucous fibrosis (OSMF), which predominantly affects the people of South-East Asian origin (Gupta et al, 1989; Cheng et al, 2004; Lalli et al, 2008). Hence, it would be of great aid if effective diagnostic/screening marker that monitors premalignant lesion were available for routine use. Many studies have focused on discovering a biomarker for screening OSCC in saliva, so as to develop a noninvasive and simple method of screening. Although several markers have been discovered in saliva in the recent years (Liu and Duan, 2012), their validity for routine use as a marker has not been much focused. Among the various salivary biomarkers of OSCC, cytokeratin fraction 21-1 (CYFRA 21-1) has been reported to be potent marker (Nagler et al, 2006). Cyfra 21-1 is a well-accepted tumor marker with high sensitivity and specificity in nonsmall cell lung cancer, especially squamous cell carcinoma (independent prognostic factor) (Nisman et al, 1998;
Salivary CYFRA 21-1- a marker for oral cancer
K Rajkumar et al
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Kawaguchi et al, 2000). While there are reports that CYFRA 21-1 is a useful oral tumor marker (Nagler, 2009), to our knowledge, the clinical utility of this marker in saliva as a screening tool in potentially malignant condition to detect OSCC has not been addressed. In this study, we have validated the diagnostic utility of CYFRA 21-1 in differential diagnosis of premalignant (leukoplakia & OSMF) and malignant oral condition (OSCC) in serum and saliva. Furthermore, the study also compared the levels of CYFRA 21-1 with histological gradation and cancer staging in patients with OSCC.
Materials and methods This study was approved by the Scientific Advisory Committee and Institutional Ethical Committee of SRM University, Chennai. According to ethical principles, including the World Medical Association Declaration of Helsinki (version, 2002), a written and informed consent was obtained from all the study participants before drawing blood and collecting saliva. Study subjects recruitment The recruitment of study subjects was carried out at outpatient department of SRM dental college, referrals from SRM General Hospital and private dental clinics, Chennai during January 2009 and December 2012. The demographic details and information on previous history were collected. Subjects with infectious diseases during 1 month before saliva sampling, active dental abscesses, systemic illness, and collagen vascular diseases were excluded from the study. None of the lesions had been treated in any manner prior to sample collection. None of the control participants had oral lesions. Pregnant and lactating subjects were also excluded. After registering the eligible patients, the histopathological examination was carried out for premalignant and OSCC subjects. A standard tissue biopsy was taken from each patient. The diagnosis was confirmed by two pathologists independently. The biopsies were further classified based on histopathological gradation. Sample collection Saliva samples were collected between 9 and 11 A.M under non-stimulatory condition. Participants were asked to refrain from eating, chewing, and drinking at least one hour before collection. 3–5 ml of salivary samples from patients with oral carcinoma and premalignant lesion was collected before any therapeutic procedure. Following collection, the saliva was immediately centrifuged to remove cell debris. Supernatants were then stored at 80°C. 2 ml of peripheral blood was drawn from all study subjects through venipuncture. Blood was transferred to an empty vacutainer. Serum was then collected by centrifuging the coagulated blood and stored at 80°C until further use. Estimation of CYFRA 21-1 Concentrations of salivary and serum CYFRA 21-1 were quantified by commercially available ELISA Kit (Syntron Bioresearch In., Carlsbad, CA, USA). The assay was
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carried out according to the manufacturer’s instruction. Absorbency was 620 nm and measured by microplate reader (RoboniK ELISA plate reader). The detection range of the kit was from a minimum of 0.5 ng to a maximum of 50 ng ml 1. The results were expressed as ng ml 1 of saliva or serum. Statistical analysis The data were analyzed using SPSS software, version 21.0 (SPSS, Chicago, IL, USA). Nonparametric Mann– Whitney U-tests were performed to find the significance of the observed differences in OSCC and PML groups. A P value of 10.4, salivary CYFRA 21-1 shows 75% specificity and sensitivity in differentiating PML from OSCC. On the other hand, a cutoff value of 2.5 ng ml 1 in serum CYFRA 21-1 level showed 90% specificity, while the sensitivity was reduced to 60%. Nevertheless, with salivary CYFRA 21-1, we evaluated cutoff value with 95% specificity in healthy control and found it to be >7.91. With this cutoff, the sensitivity was found to be
Figure 2 The levels of CYFRA 21-1 compared between serum and saliva of healthy control, PML and OSCC subjects. The results are expressed as mean SEM. The statistical significance is shown as * when compared within group, # when compared with healthy control group, and $ when compared with PML group. P < 0.05 was considered to be statistically significant
83.6% in detecting the OSCC group. Curve comparing the diagnostic ability of salivary and serum CYFRA 21-1 in detecting OSCC using ROC curve is illustrated (Figure 3). A significant difference (P < 0.05) was noted between the Oral Diseases
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Table 2 Level of serum and salivary CYFRA 21.1 levels in different study groups
Study subjects Healthy Control (n = 100) Premalignant lesion subjects (n = 100) Oral squamous cell carcinoma (n = 100)
Serum CYFRA 21.1 (ng ml 1)
Salivary CYFRA 21.1 (ng ml 1)
1.07 0.073 1.50 0.08a
3.06 0.25c 8.15 0.50ac
5.14 0.38ab
17.46 1.46abc
The levels of serum and salivary CYFRA 21.1 were assessed by ELISA in all the study subjects. The concentrations are expressed in nanogram per milliliter, and the values represent mean SEM. a P < 0.05 when compared to healthy control group, bwhen compared to premalignant lesion group, and cwhen compared between serum saliva.
AUC for salivary (0.899) and serum CYFRA 21-1 (0.865) based on pairwise comparison of ROC curves. ROC analysis demonstrates that salivary CYFRA- 21-1 was superior to serum CYFRA 21-1, specifically in differentially diagnosing OSCC with that of PML. CYFRA 21-1 levels based on histological grading and clinical staging For 100 patients with OSCC, CYFRA 21-1 concentrations were compared according to histological differentiation (Table 1, Figure 4a,b). The median and interquartile range of serum CYFRA 21-1 concentration were 4.08 (0.9– 20.4), 3.3 (0.41–19.3) and 5.6 (0.32–14.5) ng ml 1 for well-differentiated (WD), moderately differentiated (MD), and poorly differentiated (PD) lesions, respectively (Figure 4a). The serum CYFRA 21-1 concentration did not show any significant difference between the histological grading in OSCC. Median and interquartile range of salivary CYFRA 21-1 concentrations were 11.8 (6.8–28.4) for
WD, 13.9(8.2–89.2) for MD, and 18.3 (9.7–110.3) for PD lesion, respectively (Figure 4b). An overall tendency toward significant (P < 0.001) increase in salivary CYFRA 21-1 concentration was observed for PD lesion. No significant difference was evident for salivary CYFRA 21-1 concentration between WD and MD lesion. Also, no significant difference with salivary CYFRA 21-1 was found depending on the histological grading in PML (data not shown). The level of CYFRA 21-1 in serum and saliva was also compared based on the clinical staging of OSCC patients (Figure 4c,d). In serum, the median and interquartile range for stage I is 2.60 (0.32–6.40), stage II is 3.56 (0.41– 6.19), stage III is 6.97 (3.97–19.3), and stage IV is 9.29 (4.19–20.49). Similarly for saliva, the median and interquartile range for stage I is 15.35 (3.56–24.95), stage II is 14.5 (0.31–29.45), stage III is 16.4 (9.2–30.34), and stage IV is 29.5 (18.35–110.3), respectively. Interestingly, serum CYFRA 21-1 levels were significantly increased in stage III and IV (P < 0.01). No difference was observed between stage I and stage II and between stage III and IV. On the other hand, in saliva, significant increase was found only with stage IV (P < 0.001).
Discussion This is the first study comparing the use of CYFRA 21-1 as a biomarker in saliva for differential diagnosis of premalignant and malignant oral carcinoma in a population with high incidence of oral cancer. Although both serum and salivary CYFRA 21-1 levels were significantly higher for OSCC compared with PML, salivary CYFRA 21-1 showed threefold increase compared with serum CYFRA 21-1. Furthermore, the validation of CYFRA 21-1 levels
Figure 3 ROC curve analysis of CYFRA 21-1 marker in saliva and serum for predicting OSCC when compared to PML Oral Diseases
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(a)
(c)
25
* (P * < 0.001) (P < 0.001)
15
ng ml–1
ng ml–1
20
10 5 0
(b)
(P < 0.001)
(P < 0.01)
Stage I
Stage II
Stage III
Stage IV
(d)
100
(P * < 0.001)
ng ml–1
ng ml–1
80 60 40 20
0
Stage I
Stage II
Stage III
Stage IV
Figure 4 The levels of CYFRA 21-1 compared in serum and saliva of OSCC subjects based on histological grading (a and b) [well differentiated (WD), moderately differentiated (MD), and poorly differentiated (PD)] and based on clinical staging (c and d). Each dot represents CYFRA 21-1 level of each individual subject, and the horizontal line represents mean value. The statistical significance is shown as * when compared to healthy control and # when compared to PML subjects. P < 0.05 was considered to be statistically significant
with ROC curve revealed salivary CYFRA 21-1 to have high sensitivity for OSCC. CYFRA 21-1 is a well-established monoclonal antibody that reacts exclusively with cytokeratin-19. Cytokeratin family consists of 19 different polypeptides, which have been numbered 1–19 (Moll et al, 1982). These cytokeratins are not randomly distributed in epithelia, but appear to be characteristic for certain types of epithelial differentiation (Gasparoni et al, 2005). Cytokeratin-19 is an acidic (type I) subunit expressed in all simple epithelia and in carcinomas which arise from them (Broers et al, 1985, 1988), Hence, it was found to be a potential marker in many squamous cell carcinomas. CYFRA 21-1 has been reported to be an established marker for lung cancer (Nisman et al, 1998). In addition, it is also shown to be a potent marker for head and neck cancer, gastric cancer, uterine cancer, and esophageal cancer (Doweck et al, 1995; Nakata et al, 1996; Bonfrer et al, 1997; Yamamoto et al, 1997). The utility of CYFRA 21-1 for OSCC was first demonstrated by Kurokawa et al (1997). They showed significant increase in serum CYFRA 21-1 of patients with OSCC compared with control and benign oral tumor subjects. Later many studies reported high levels of serum CYFRA 21-1 in OSCC with varying sensitiv-
ity and specificity. Some studies claimed serum CYFRA 21-1 to be poor diagnostic marker in detecting OSCC because of low sensitivity and specificity (Hamakawa et al, 1998; Yen et al, 1998; Feng et al, 2010), while other studies showed it to be accurate diagnostic and prognostic marker for OSCC (Nagler et al, 2006; Zhong et al, 2007b). This discrepancy may be due to the difference in the methodology employed in measuring CYFRA 21-1 and study design. In accordance with Nagler et al (2006) and Zhong et al (2007a), we also found higher levels of serum CYFRA 21-1 in OSCC subjects compared with controls. According to Sugama et al (1994), increased serum CYFRA 21-1 is the net result of cytokeratin release due to cell lysis or necrosis. Moreover, during epithelial transition to malignancy, there is an increase in the degradation of cytokeratin which in turn results in release of large amount of cytokeratin fragments in the local environment (Ausch et al, 2010). Taking this into account, it is more likely for saliva, the local environment of OSCC to show high levels of CYFRA 21-1. In concordance with this hypothesis, Nagler et al (2006) found fourfold increase in amount of CYFRA 21-1 in saliva compared with serum illustrating saliva to be best medium for OSCC diagnosis Oral Diseases
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(Nagler et al, 2006). This was again proved with another study where salivary CYFRA 21-1 levels were significantly higher in OSCC subjects (Zhong et al, 2007a). Concordant to the above reports, our study showed threefold higher level of CYFRA 21-1 in saliva when compared to serum. From the present study, we also find significantly higher CYFRA 21-1 levels in saliva of PML and not in serum compared with control. This shows that there occurs some change in these oral lesions, which is immediately reflected in the saliva rather in serum. The ROC curve analyses further confirmed salivary CYFRA 21-1 to possess higher sensitivity and specificity than serum CYFRA 21-1. Although the specificity of serum CYFRA 21-1 was more than 85%, their sensitivity was low to be considered for diagnostic utility. Moreover, high sensitivity is the hallmark for a marker to be used for screening test, from the present study it is evident that salivary CYFRA 21-1 is more useful as a screening marker rather than serum CYFRA 21-1 levels. Also, we found salivary level to be significantly higher with poorly differentiated lesions of OSCC, but the same was not reflected in serum levels. However, when compared based on clinical staging of cancer, we found CYFRA 21-1 to be increased in stage IV in serum and saliva. This shows that CYFRA 21-1 levels are more increased toward the end stage of cancer, and therefore, CYFRA 21-1 level is shown to increase with the severity of disease as reported by others (Brockmann et al, 2000). Of late, reports show that there is decline in serum CYFRA 21-1 after chemoradiotherapy in esophageal squamous cell carcinoma, suggesting it as a prognostic marker (Yi et al, 2009; Yan et al, 2012). Although it is well established that radiation affects salivary flow rate and secretion, it would be of interest to know the status of salivary CYFRA 21-1 in all types of OSCC, so that it can be considered for prognostic marker as well. The main limitation of this study is that it did not include other PML lesion such as erythroplakia and oral lichen planus in PML group. The reason for their exclusion is their rare occurrence, and moreover, leukoplakia and OSMF are the one which are stated to transit to malignant form at a high rate compared with others. So, this should not undervalue the outcome of the study. It is also important to note the effect that tobacco smoking/ chewing has on CYFRA 21-1 levels. But it could not account for the difference in CYFRA 21-1 levels between the patients with cancer and the PML subjects, as the numbers of smokers among these two groups were similar, and moreover, it is reported that smoking has no effect on CYFRA 21-1 concentration (Kao et al, 1999). Therefore, in healthy smokers also, it should not affect the salivary or serum CYFRA 21-1 levels. In summary, the significantly higher levels of salivary CYFRA 21-1 in OSCC is encouraging in light of the many advantages of saliva measurement in comparison with serum analysis. This suggests that salivary CYFRA 21-1 may be considered for diagnostic tool as it is often very difficult to distinguish clinically between a premalignant and OSCC by visual screening alone. Accordingly, such an analysis may turn into a valuable screening tool as it might save many unnecessary biopsies and will also
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allow easy adherence of patients for constant follow-up for screening in regions with high incidence of PML due to tobacco intake. Acknowledgements The authors thank the patients and healthy volunteers in the study for their willingness to contribute to medical research. The statistical suggestion by Dr.C. Ponnuraja (Biostatistics Department, National institute of Research in Tuberculosis) is acknowledged.
Author contributions K. Rajkumar conceived and designed the study, interpreted the data and revised the manuscript. R. Ramya was involved in design, sample collection and data interpretation, G. Nandhini did sample collection, carried out ELISA, data management, P. Rajashree did data analysis, statistics, interpretation and manuscript writing, K. A. Ramesh contributed to sample collection and A. S. Nirmala was involved in interpretation and revision of manuscript.
Conflict of interest The authors declare no conflict of interests.
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