Do patients with oral squamous cell carcinoma express receptor tyrosine kinase-like orphan receptor 1? Results of an observational study.

CONTEXT: The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transmembrane protein of the receptor tyrosine kinase family. The expression of ROR1 has been linked to cancers. AIMS: This study aimed to investigate the expression of ROR1 in oral squamous cell carcinoma (OSCC). SETTINGS AND
DESIGN: This prospective observational study was conducted at a tertiary referral center for treatment of oral carcinoma from November 2013 to December 2016. SUBJECTS AND METHODS: One-step quantitative reverse transcription-polymerase chain reaction (30 oral cancer tissues and ten normal oral tissue samples) was performed to characterize the expression of the ROR1 gene in oral cancer. STATISTICAL ANALYSIS USED: Analyses of all tumor samples were carried out at least twice, and the mean value was calculated. The differences in ROR1 messenger RNA (mRNA) expression between OSCC tissue and nontumorous gingival tissue was statistically analyzed using Mann-Whitney U-test. The correlations between the clinicopathological parameters and ROR1 mRNA expression were analyzed using Kruskal-Wallis test χ2 value.
RESULTS: There were 17, 5, 3 and 1 cases of OSCC of buccal mucosa, tongue and lower alveolus lip, respectively. Nearly 88.5% of cases had a history of tobacco consumption. The most common OSCC type was T2N1M0. There was no difference in ROR1 fold change between controls and cases (P = 0.06), but there was a trend for downregulation of ROR1 expression from controls to cases. Subgroup analysis revealed the downregulation of ROR1 expression in controls versus Grade II that was significant (P = 0.04).
CONCLUSIONS: There was no change in the expression of ROR1 between cases and controls. A study involving a larger sample size needs to be formulated and conducted for investigating the relation between expression and regulation of ROR1 in OSCC. Copyright:

INTRODUCTION

Oral cavity is one of the ten frequent sites of cancer.[1] Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that participate in crucial cellular processes. These processes include cell proliferation, differentiation, cell–cell interaction, metabolism, signaling, migration and survival.[2] Recently, studies have linked one of the RTKs, RTK-like orphan receptor 1 (ROR1), with different cancers. ROR1 has been extensively studied in hematological malignancies.[345678] To the best of our knowledge, the expression of ROR1 in oral carcinoma has not been studied. We analyzed the expression patterns of ROR1 in malignant and nonmalignant oral tissues using real-time quantitative polymerase chain reaction (qPCR).

Aims and objectives

The objectives of this study were:

To assess and compare the quantitative expression of ROR1 in oral squamous cell carcinoma (OSCC) and controls

To compare the expression of ROR1 in different histological grades of OSCC.

SUBJECTS AND METHODS

This prospective case–control study was conducted at a tertiary referral center for the treatment of cancers from November 2013 to December 2016. We collected tissue samples from thirty consecutive patients, which were operated for OSCC. Ten samples of normal gingival tissue (controls) were collected from patients presenting to the outpatient department for other reasons. All the samples were collected in RNA later (Thermo Fisher Scientific Waltham, MA USA) from the operating room, transported (at −4° centigrade©) and stored at −20°C. The samples were subjected to qPCR [Figure 1].

Figure 1

Summary of steps in quantitative PCR

The total RNA was extracted by RNA extraction kit. Quantification of extracted total RNA was done by spectrophotometer and expressed in ng/μl

Quality and purity of RNA was tested by UV spectrophotometer. The ratio of absorbance at 260 and 280 nm on spectrophotometer was measured, and the ratio above 2 was considered high-quality RNA. The samples were subjected to gel electrophoresis to find out the quality of RNA. The samples not forming any band on gel electrophoresis were excluded from further analysis

The complimentary DNA (cDNA) synthesis was done from the extracted RNA template using reverse transcriptase. One nonreverse transcriptase was used with all samples tested to prevent false-positive outcomes

Before proceeding for qPCR, primers were validated at different annealing temperatures, concentrations and cDNA volume. Annealing temperatures of 52.1°C, primer concentration of 0.5 μMol and cDNA volume of 0.75 μl were selected for qPCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was taken as a housekeeping gene. The ratio of ROR1 to GAPDH represented the normalized relative levels of ROR1 expression. A nontemplate negative control was also included in each experiment

The amplification of DNA was done using steps of denaturation, annealing and extension at 94°C for 2 min, 56°C for 30 s and 72°C for 15 s, respectively. Forty-four such cycles were run to get amplified DNA. Figure 2 shows sigmoid-shaped amplification plot of the amplified DNA. Here, cycle threshold (CT) denotes the cycle number at which fluorescence generated by the PCR product (amplified DNA) of a given sample is distinguishable from the background noise. The difference of CT of ROR1 and GAPDH of a particular sample of either a case or control was called Delta CT, which was given by the formula

Figure 2

Amplification plot of the DNA

Delta CT= CT(ROR1)-CT(GAPDH)

An analysis of all samples of cases and controls was carried out at least twice, and the mean value was calculated, which was called average Delta CT. Delta Delta CT was obtained from Delta CT by formula: Delta Delta CT= average delta CT(sample of interest)-average delta CT(reference sample)

The reference sample was one, which was having the matched pair of Delta CT values in duplicates

Relative fold change expression of ROR1 gene was calculated by (Statistical Package for the Social Sciences) SPSS software

Fold gene expression = 2−ΔΔCT

Values were expressed as fold change in expression of ROR1 gene compared to GAPDH. Demographic data, clinicopathologic findings, and results of PCR were recorded. The differences in ROR1 messenger RNA (mRNA) expression between OSCC tissue and nontumorous gingival tissue were statistically analyzed using Mann–Whitney U-test. The correlations between the clinicopathological parameters and ROR1 mRNA expression were analyzed using Kruskal–Wallis Test X2 value. All statistical calculations were carried out using SPSS software (for Windows, Version 20; Chicago Inc., USA). A significant difference was defined at P ≤ 0.05.

RESULTS

A total of 36 participants were included for analysis through real-time PCR. Four samples from OSCC cases were found inadequate and excluded at the final evaluation. There were 26 cases and ten controls. The mean age of the patients was 47.96 years for cases and 40.0 years for controls. The demographic variables in cases and controls are described in Table 1. The most common site for involvement was buccal mucosa (65.4%). The cases were classified as per TNM classification by imaging, biopsy and Preoperative findings [Table 1]. The most common type was T2N1M0. Around 88.5% of the cases were either tobacco chewers or smokers. Nearly 19.2% of the cases were alcoholics.

Table 1

Demographic data between cases and controls

Parameter	Cases (%)	Controls (%)	P (<0.05)
n	26	10
Age ( years)
30-40	9 (34.6)	7 (70.0)
41-50	6 (23.1)	2 (20.0)
51-60	6 (23.1)	1 (10.0)
61-70	5 (19.2)	0 (0.0)
Gender
Males	23 (88.5)	8 (80.0)
Females	3 (11.5)	2 (20.0)
Site
Buccal mucosa	17 (65.4)
Tongue	5 (19.2)
Lower alveolus	3 (11.5)
Lip	1 (3.8)
Tobacco consumption	23 (88.5)		0.00
Smoking	9 (34.6)		0.032
Alcohol consumption	5 (19.2)		0.135
Tumor size, node and metastasis
T1	1 (3.8)
T2	13 (50)
T3	4 (15.4)
T4	8 (30.8)
N0	10 (38.5)
N1	11 (42.3)
N2	5 (19.2)
M0	26 (100)

The mean weight of the tissue for analysis of qPCR from cases and controls was 30–35 mg. The mean value of total RNA extracted using kit was 187.89 ± 30.95 ng/μl for cases and 105.09 ± 64.48 ng/μl. The quantitative analysis of ROR1 was measured in overall; there was a trend for downregulation in ROR1 expression from controls to cases, which failed to reach statistical significance. However, subgroup analysis showed downregulation in ROR1 expression between Grade II and control which was significant [Table 2]. There was no difference in ROR1 expression between Grade I and III with controls. The analysis of fold change in expression of ROR1 between different grades, tumor size and nodal metastasis was also done, which is given in Table 2. Neither tumor size T1 to T4 (P = 0.237) nor nodal metastasis (N0 to N2) (0.32) demonstrated any difference in fold change in ROR1 expression. Comparison of fold change in ROR1 expression among tobacco consumers and nontobacco consumers did not demonstrate any difference between the groups (P = 0.42).

Table 2

Table demonstrating median receptor tyrosine kinase-like orphan receptor 1 fold change between cases/controls and histopathological grades for different subgroups

Outcome	ROR1 fold change median (IQR)	P
Cases/controls	0.19 (0.06-0.80)/0.81 (0.23-1.41)	0.06
Grade I/controls	0.25 (0.04-1.20)/0.81 (0.23-1.41)	0.26
Grade II/controls	0.19 (0.08-0.23)/0.81 (0.23-1.41)	0.04
Grade III/controls	0.24 (0.03-0.24)/0.81 (0.23-1.41)	0.27
Grade I/Grade II	0.25 (0.04-1.20)/0.19 (0.08-0.23)	0.89
Grade II/Grade III	0.19 (0.08-0.23)/0.24 (0.03-0.24)	0.91
Grade I/Grade III	0.25 (0.04-1.20)/0.24 (0.03-0.24)	0.70

ROR1: Receptor tyrosine kinase-like orphan receptor 1, IQR: Interquartile range

DISCUSSION

ROR1 is a embryonal transmembrane glycoprotein, but studies have demonstrated its presence in some adult tissue such as testis, uterus, lung, bladder, colon, pancreas and adipose tissue.[3] Recently, the expression of ROR1 has been linked with solid tumors.[9101112131415]

Our study did not demonstrate any difference in ROR1 fold change between controls and cases, but there was a trend for the downregulation of ROR1 expression from controls to cases. Subgroup analysis revealed the downregulation of ROR1 expression in controls versus Grade II that was significant. Liu et al. studied ROR1 expression in squamous cell carcinoma of lung.[12] The investigators reported downregulation of ROR1 expression in cases of squamous cell carcinoma of lung. Although the results of our study failed to reach statistical significance, there was a downregulation of ROR1 expression in cases. Studies have demonstrated ROR1 as a prognostic factor for different tumors (breast cancer and colorectal cancer).[1115] ROR1 has been linked with tumor aggression across several studies. The linking of ROR1 has been studied for chronic lymphoid leukemia, acute lymphoid leukemia, lung adenocarcinoma, ovarian cancer, melanoma and glioblastoma.[3468910121314] The results of our study suggest that the lower value of the ROR1 expression is indicative of the poor prognosis of patients with OSCC that can be investigated further by studies with a larger sample size. ROR1 might be acting as a tumor suppressor gene. Although the role of ROR1 in cancers is not well understood, it may be governed by the Wnt5A-dependent inhibition of canonical Wnt signaling downstream of beta-catenin stabilization, thereby inhibiting tumor cell growth, inducing cell cycle arrest and apoptosis. Our study is the first on the estimation of ROR1 expression in OSCC. In our study, real-time PCR was performed with optimized values. qPCR is one of the modern methods for studying gene expression. We used sequence-specific DNA probes with SYBR GREEN fluorescent dye for the optimization of results. One of the limitations of our study is the small sample size of thirty patients of OSCC. Given the incidence of OSCC worldwide, studies with larger sample numbers are required to analyze the role of ROR1 in OSSC. Other methods for the estimation of gene expression such as immunohistochemistry, DNA microarray and Western blotting can be combined with qPCR for increasing the precision of results. Despite the substantial advances in surgery and chemotherapy for oral cancer over the past few decades, therapeutic failure and disease progression are still frequent. For several other cancers such as breast cancer, ovarian cancer and CLL, ROR1 has been studied extensively and found as a biomarker for targeted therapy. This protein also served as an appropriate candidate for designing a cancer vaccine. Thus, we have been searching a novel and valuable marker that associates with oral cancer and could be used as a potential protein for the treatment of oral cancer.

CONCLUSION:

There was no change in expression of ROR1 between cases and controls. A study with larger sample size needs to be conducted for investigating the expression of ROR 1 in OSCC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.