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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 3  |  Page : 452-457

Biochemical analysis of cytokeratin fragment 21-1 concentration and expression of cytokeratin 19 in oral potentially malignant disorders


Department of Oral and Maxillofacial Pathology and Microbiology, ITS-CDSR, Muradnagar, Ghaziabad, Uttar Pradesh, India

Date of Submission24-Oct-2017
Date of Decision23-Jan-2018
Date of Acceptance26-Feb-2018
Date of Web Publication26-Oct-2018

Correspondence Address:
Sonal Katyal
ITS-CDSR, Muradnagar, Ghaziabad, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_893_17

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 > Abstract 


Introduction: Cytokeratin fragment 21-1 (CYFRA21-1), a constituent of the intermediate filament protein is known to be elevated in cancer. In vitro cleavage of cytokeratin 19 (CK19) protein results in the release of it's fragments into the supernatants of premalignant cell lines. This study was designed with the aim to investigate the concentrations of CYFRA21-1 in serum and saliva of oral potentially malignant disorders (OPMD), to evaluate CK19 expression in tissues of the same patients and to correlate the levels of CYFRA21-1 concentration in serum and saliva with CK19 expression in OPMDs, and to compare it with oral squamous cell carcinoma (OSCC), which was taken as positive control.
Materials and Methods: Concentration of CYFRA21-1 was measured in saliva and serum of 30 OPMD cases with five patients having OSCC using ELISA technique and analysis of CK19 protein expression in the tissue of same patients using immunohistochemical technique was done.
Results: Concentration of CYFRA21-1 in saliva and serum with regard to CK19 protein expression in tissues was significantly higher in control group than in study groups.
Conclusion: CYFRA21-1 can be used as a promising diagnostic molecule and as an adjunctive marker for early detection, disease staging, and monitoring.

Keywords: Cytokeratins 19, cytokeratin fragment 21-1, ELISA, immunohistochemistry, saliva, serum


How to cite this article:
Rathore AS, Katyal S, Jain A, Shetty DC. Biochemical analysis of cytokeratin fragment 21-1 concentration and expression of cytokeratin 19 in oral potentially malignant disorders. J Can Res Ther 2020;16:452-7

How to cite this URL:
Rathore AS, Katyal S, Jain A, Shetty DC. Biochemical analysis of cytokeratin fragment 21-1 concentration and expression of cytokeratin 19 in oral potentially malignant disorders. J Can Res Ther [serial online] 2020 [cited 2020 Aug 7];16:452-7. Available from: http://www.cancerjournal.net/text.asp?2020/16/3/452/244239




 > Introduction Top


Oral squamous cell carcinoma (OSCC) carcinogenesis involves the accumulation of genetic alterations that coincide with the multistep malignant transformation of the normal oral epithelium. OSCC is often diagnosed at late stages of the disease (advanced regional disease and/or metastasis) and the 5 year survival rate, i.e., 50%–55% for oral cancer has not improved significantly. To date, early detection, diagnosis, and treatment remain the key points to determine the malignant conversion of the potentially malignant disorder.[1],[2]

The term, potentially malignant disorders, was recommended to “precancer” as it conveys that not all disorders described under this term may transform into cancer. The WHO Collaborating Centre for Oral Precancerous Lesions in 1978 sought to define Oral Leukoplakia, Oral Lichen Planus, and Oral Submucous Fibrosis sufficient in tightly to providing an internationally accepted system that carried an increased risk of potentially malignant lesions.[3]

The use of noninvasive genetic tests or molecular markers in saliva and serum proved to be a promising relevant prognostic marker in cases of early detection,[4] such as cytokeratins (CKs), carcinoembryonic antigen, squamous cell carcinoma antigen, and immunosuppressive protein.[5],[6],[7]

Studies have shown that CYFRA21-1 is a soluble fragment of CK19 and is extremely sensitive and specific marker, providing a valuable prognostic pointer for the detection of continuing disease and also for the evaluation of response to treatment.

CK19 is an acid type CK with a molecular weight of 40 kDa, having an isoelectric pH = 5.2.[8],[9],[10]In vitro cleavage of CK19 protein occurs through spontaneous caspase 3 activity, resulting in the release of CYFRA21-1 into the supernatants of cancer cell lines.[11],[12],[13]

According to the literature review, there is a paucity of studies using CYFRA21-1 as a prognostic biomarker in serological assays with CK19 expression in oral potentially malignant disorders (OPMDs). Therefore, this study was designed to investigate the saliva and serum CYFRA21-1 concentration in OPMDs as well as CK19 expression in lesional tissues of the same patients using ELISA and immunohistochemistry, respectively.


 > Materials and Methods Top


Patients and samples

The study sample comprised 30 patients of OPMDs reported to the Department of Oral Pathology and Microbiology. Totally 10 patients each of leukoplakia, lichen planus, and oral submucous fibrosis (OSMF) and five cases of OSCC as positive control were taken. Ethical clearance was obtained from the Institutional Ethical Committee. Saliva samples, serum sample of all 35 patients were taken with informed written consent. Of these 35 patients, there were 24 males and 11 females, age ranging between 38 and 52 years.

Method of sample collection

Saliva samples of all 35 patients were collected before any medical treatment. The prior collection, gargling was advised with a sterile saline solution to clean the oral cavity. A volume of 3 ml unstimulated saliva was collected into a sterile test tube using the drooling method. The saliva was centrifuged at 3300 rpm for 10 min. The supernatant was used for detection.

A volume of 5 ml of venous blood sample was collected from all 35 patients in yellow top silica coated vacuotainers. The samples were allowed to clot for 1 h and then centrifuged at room temperature and the supernatant was collected. The supernatant of saliva and serum were poured into tubes, sealed, and stored at −20°C until processing.

Incisional biopsy was performed and tissue sample of the lesion was obtained from the same patients. Samples were fixed in 10% neutralized formalin, processed and then embedded in paraffin for use. Sections were used for histological examination including hematoxylin and eosin (HE) and for immunohistochemical detection of CK19 protein

Detection of cytokeratin fragment 21-1 concentration in serum and saliva by ELISA

CYFRA21-1 concentration was measured using the CYFRA21-1 ELISA kit forIn vitro diagnostic use, containing 96 wells (Immunoconcept, India) according to the manufacturer's instruction. First in each microtiter plate well, 50 μl of Calibrators Cal 1–5 and unknown samples were added, 50 μl of CONJ HRP was dispensed on the samples in order, the wells were mixed gently for 30 s. The plate was sealed and incubated for 60 min at 37° C, then washed 5 times using buffer wash ×21 followed by addition of 100 μl SUBS TMB and then again incubate 10–20 min at 18°C–25°C. Finally, the reaction was stopped by adding 100 μl STOP, the wells were gently mixed for 30 s, and optical density of each microtiter plate well was read by microtiter plate reader at 450 nm within 30 min.

The CYFRA21-1 standard five samples (negative and positive controls) from the kit, with the standard CYFRA21-1 concentration of 0, 3.8l, 10l, 25, and 50 ng/dl, were added into the wells to plot the standard curve. The CYFRA21-1 concentration of each sample was determined from the standard curve. If the CYFRA21-1 concentration was more than 50 ng/dl, the samples were diluted and then detected again until the concentration was <50 ng/dl.

Detection of cytokeratin 19 protein expression in cancerous and potentially malignant tissues by immunohistochemistry

Serially cut sections, 4–5 μm thick, were made from each paraffin block with a rotary microtome and collected on polyvinyl coated slides. Each sample was studied by HE and immunohistochemical staining for CK19. HE-stained sections were reviewed according to the WHO histologic criteria.[14]

After deparaffinization with xylene, sections were transferred to water through ethanol.

Before incubating in antibody solutions, sections were heated by water bath at 98°C with 0.01 M citrate buffer solution (pH = 6.0) for 20 min to retrieve antigen, cooled at room temperature, and washed with phosphate buffer solution (PBS) three times for 5 min each.

Sections were then mixed with antibody solutions in the following order: 3% hydrogen peroxide once for 5 min, PBS three times for 2 min each, mouse monoclonal antibody to CK19 (dilution: 1/50) (Antibody Biogenex, India) for 1 h, PBS three times for 5 min each then super enhancer and SS label 25 min each, PBS three times for 5 min each. The reactions were carried out in a moist box at room temperature. After incubation in the antibody solutions, reaction results were visualized by 3,3 diaminobenzidine detection kit (Biogenix, India). Finally, sections were washed in distilled water, counterstained by hematoxylin for 2 min, washed with tap water and ethanol, and covered with coverslips. Negative controls were prepared by PBS instead of the primary antibody.

Immunohistochemically stained slides of OPMD's and OSCC were viewed by two pathologists. A blind study was performed. CK19 showed positive cytoplasmic staining in few cells of OPMD's and OSCC's. The staining intensity was scored on a scale of negative to strong as, Score 0-Negative (0% of reactive cells), Score 1-Weak (1%–25% of reactive cells); Score 2-Moderate (26%–50% of reactive cells); Score 3-Strong (>50% of reactive cells). The expression of marker was assessed as follows: 0 - no staining or staining in <10% of the tumor cells; 1 - staining in 10%–25% of the cells; 2 - staining in 26%–50% of the cells; 3 - staining in 51%–75% of the cells; 4 - staining in more than 75% of the cells.

The expression index of CK19 was calculated by the below-mentioned formula, to acquire the median value:

Expression index = Percentage positivity of cells × intensity score

Statistical analysis

All data were analyzed using the statistical software of SPSS version 16.0 for Windows. The normality of the concentration of CYFRA21-1 in serum and saliva was tested by Shapiro Wilk Test, and we found that data were normally distributed for all the groups. The significant difference of mean concentrations of CYFRA21-1 in serum and saliva between the groups was tested using one-way ANOVA followed by the Bonferroni test. The correlation between the concentration of serum and saliva with CK19 immunohistochemical score was calculated by Spearman's Rank Correlation. The comparative analysis was performed using paired t-test. When the value of P < 0.05, the difference was considered as statistically significant. The level of significance was 5% and 1%.


 > Results Top


Cytokeratin fragment 21-1 concentration in saliva and serum of oral potentially malignant disorder groups

The concentration of CYFRA21-1 in saliva and serum of various study groups and control group was analyzed using the standard plotted graph of the five calibrations. The mean and standard deviation of the concentration of CYFRA21-1 in serum and saliva in study groups on comparison showed maximum value in OSMF and least in Lichen Planus in serum. Similarly, in saliva also Lichen Planus had the least value, i.e., 22.93 ± 2.64 in comparison to OSMF, i.e., 31.52 ± 2.28 [Table 1]. Salivary and serum CYFRA21-1 concentration within groups when correlated showed statistically significant results. Furthermore, in control group, i.e., OSCC, both saliva and serum concentration values were significantly higher (P = 0.01) than study groups.
Table 1: Comparison of CYFRA 21-1 concentration in serum and saliva in study groups

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The difference in concentration of CYFRA21-1 in saliva, as well as serum, was significant between the control group and various study groups [Table 2].
Table 2: Comparison of concentration of CYFRA 21-1 in saliva and serum within groups

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Wide variation in the concentration of CYFRA-21 in saliva and serum of OPMD's and OSCC (Positive Control Group) patients was noted. The mean concentration of CYFRA21-1 in serum was 12.3, 16.2, 20 and 34 ng/dl in Lichen Planus, Leukoplakia, OSMF, and OSCC, respectively; whereas the concentration of CYFRA in saliva was significantly higher in the same groups, i.e., 26, 32, 33, and 50 ng/dl [Figure 1]a and significantly increased the concentration of CYFRA21-1 was noted in saliva samples [Figure 1]b.
Figure 1: Graphical representation of (a) cytokeratin fragment 21-1 concentration in oral potentially malignant test groups; (b) Increased concentration of cytokeratin fragment 21-1 in saliva as compared to serum

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Cytokeratins 19 protein expression in cancerous and potentially malignant tissues

In OPMDs, faint expression of CK19 was seen in the basal layer of mucosa [Figure 2], and negative for other cell layers of the epithelium, whereas in OSCC tissue, suprabasal layer, as well as the invasive front, was strongly positive for CK19.
Figure 2: Cytokeratin 19 protein expression in (a) oral submucous fibrosis; (b) leukoplakia; only the basal layer of epithelium is faintly positive, the suprabasal layer is negative; (c) oral squamous cell carcinoma; suprabasal layer of mucosa as well as invasive cancerous tissue was strongly positive for CK19 protein expression (immunohistochemical, ×40)

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Scoring and expression Index of CK19 in study groups was calculated and was obtained lowest in Lichen Planus and highest in OSCC.

CK19 expression score was significantly correlated with increasing concentration of CYFRA21-1 in serum and saliva of OPMD and OSCC patients [Table 3].
Table 3: Comparison of expression of ck19 in test groups

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 > Discussion Top


Saliva has been studied for many years as a promising detective media in patients with local diseases and systemic diseases.[15],[16],[17],[18],[19] Many studies have focused on discovering a biomarker in saliva and serum of OPMD's, to develop a noninvasive and simple method of screening and can be used as a prognostic tool and an additional monitoring system for early recognition of progression of the disease.

CYFRA21-1 is the saliva and serum soluble fragment of CK19 and was first described in the mid-1990s. Increased concentration of CYFRA21-1 was shown to be associated with poorer prognosis. Studies have shown CYFRA21-1 serum, and saliva levels are significantly higher in patients with Head and Neck Squamous Cell Carcinoma compared to a healthy or control group. However, very few studies have been done so far in revealing its usefulness in OPMD.

Various studies have reported the potential clinical usefulness of detecting tumor markers in OSCC and other Malignancies; a study done by Ilana Doweck et al. (1995) found serum CYFRA21-1 levels were in good correlation with tumor stage expressed by the tumor size and the lymphatic spread. Nagler et al. examined the early diagnosis and treatment monitoring roles of salivary CYFRA21-1 and found to be significant. A study by Deng et al. (2003) found that serum levels of CYFRA21-1 in patients with OSCC were significantly higher than those of healthy controls. Zhong et al. found there was a significant correlation in serum CYFRA levels with tumor recurrence and survival rate. Sheard et al. have also reported the increase of extracellular CYFRA21-1 concomitantly with an increase of intracellular CYFRA21-1 during apoptosis; in addition, the cell dying by caspase-independent death in the presence of the Z-VAD caspase inhibitor did not liberate measurable CYFRA21-1. Hence, the release of CYFRA21-1 has been suggested to arise in cells during an intermediate stage of apoptosis, on account of caspase activation, then into the extracellular space.[20],[21]

Although very less studies are available on OPMDs, where CYFRA21-1 can be used as tool for early detection, since saliva contacts directly with oral mucosa, several studies have reported the potential clinical usefulness of detecting tumor markers in saliva for OSCC, such as p53, p16, microsatellite, and telomerase activity.[8],[22],[23]

In the present study, although saliva and serum CYFRA21-1 concentrations in OSCC patients were significantly higher than those in potentially malignant disorders salivary CYFRA21-1 showed 3 times the value than serum CYFRA21-1 concentration in cases of study groups as well as in OSCC group [Figure 1]. Studies in OSCC using CYFRA21-1 assay has been suggested as a useful screening tool in suspected cases.

Furthermore, saliva CYFRA21-1concentration was more as compared to serum CYFRA21-1 concentration in various study groups [Table 2], and statistically significant results were obtained on correlation.

In OPMD tissue, CK19 protein expression is only positive in the basal layer of mucosa. In OSCC tissue, it is always positive in the suprabasal layer of mucosa as well as in invasive front of OSCC tissue.[9] However, the positive rate of CK19 expression in OSCC tissue has been reported ranging from 29% to 100%.[22],[23] It has been regarded as an excellent marker of epithelial differentiation regardless of its origin from endoderm, neuroectoderm, or germ cell lineage.[23]

Interestingly, great correlation is found between saliva CYFRA21-1 concentration, serum CYFRA21-1 concentration and tissue CK19 positive score, a higher CK19 positive score in control group tissue indicating higher saliva CYFRA21-1 concentration. [Figure 3] shows graphical representation using the linear coefficient of determination between CK19 score and concentration of CYFRA21-1 in saliva and serum.
Figure 3: Graphical representation of linear coefficient of determination between (a) CK 19 score and concentration of CYFRA 21-1 in saliva and, (b) CK 19 score and concentration CYFRA 21-1 in serum

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 > Conclusion Top


Hence, the prognostic evaluation with serum and saliva is suggested over conventional biopsy, staging, and grading using CYFRA21-1 in potentially malignant cases. However, further studies are encouraged to reveal its clinical value in predicting early detection.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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